Class of stable heptamethine cyanine fluorophores and biomedical applications thereof

ABSTRACT

Embodiments of C4′-alkyl-ether heptamethine cyanine fluorophores according to general formula I, and pharmaceutically acceptable salts thereof, are disclosed. Methods of making and using the C4′-alkyl-ether heptamethine cyanine fluorophores also are disclosed.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. patent application Ser. No.15/524,567, filed May 4, 2017, issued as U.S. Pat. No. 10,280,307, whichis the U.S. National Stage of International Application No.PCT/US2014/064136, filed Nov. 5, 2014, which was published in Englishunder PCT Article 21(2), each of which is incorporated by reference inits entirety herein.

FIELD

This disclosure concerns heptamethine cyanine fluorophores, and methodsof making and using the fluorophores.

BACKGROUND

Fluorescent small molecules are central in many modern biologicaltechniques. Their preparation often relies on inefficient condensationreactions requiring harsh reaction conditions with poor substrate scope.Thus, there is a need for new methods to prepare fluorophores for modernapplications.

Near-infrared (near-IR, 650-900 nm) fluorophores find increasing use fora variety of techniques due to the low autofluorescence in this range.Near-IR fluorophores are uniquely well suited for in vivo fluorescenceimaging due to improved tissue penetration of near-IR compared toshorter wavelengths. Heptamethine cyanines, with emission maxima around800 nm, are perhaps the archetype (Frangioni, Curr. Opin. Chem. Biol.2003, 7, 626-634; Alford et al., Molecular Imaging 2009, 8, 341-354).While useful in a variety of applications, including for certainclinical diagnostic procedures, many suffer from chemical stabilityissues at C4′ and challenging synthesis. Cyanines modified at the C4′position with an O-alkyl substituent are desirable because these arelikely to be quite stable and there is potential for a concise route tosymmetrical bioconjugatable variants. However, such molecules have onlyrarely been described and are unknown when functionalized forbiomolecule conjugation. The scarcity of C4′-O-alkyl ether cyanines isbased on the inability of most alkoxide nucleophiles to undergo thestandard preparative reaction, C4′-chloride exchange (Strekowski et al.,J. Org. Chem. 1992, 57, 4578-4580). This failure likely stems from thepoor kinetics of alkoxides in the proposed electron transfer S_(RN)1pathway, and competitive addition to the imine-like C2 position has beenreported to intercede (Strekowski et al., J. Org. Chem. 1992, 57,4578-4580; Strekowski et al., Dyes Pigments 2000, 46, 163-168). Thus, aneed exists for stable, near-IR heptamethine cyanine fluorophores and asynthetic method for making the fluorophores.

SUMMARY

This disclosure concerns a synthetic method for making cyaninefluorophores, particularly stable heptamethine cyanine (Cy7)fluorophores, embodiments of Cy7 fluorophores made by the disclosedmethod and pharmaceutical salts thereof, and methods of using the Cy7fluorophores.

Embodiments of the disclosed Cy7 fluorophores have a structure accordingto formula I

wherein m is 2, 3, 4, or 5; n is 1, 2, or 3; R¹ is —CR^(a) ₂— where eachR^(a) independently is H, halo, optionally substituted alkyl, oroptionally substituted aryl; R² is optionally substituted alkyl,optionally substituted heteroalkyl, optionally substituted aryl, oroptionally substituted heteroaryl; R³ is a maleimidyl-containing group,a succinimidyl-containing group, optionally substituted alkoxy,optionally substituted alkyl carbonyl, optionally substituted alkoxycarbonyl, a drug, or a biomolecule-containing group; R⁴ to R¹³independently are H, optionally substituted alkyl, optionallysubstituted amino, or a sulfonate-containing group, wherein R⁶ and R⁷optionally together form a substituted or unsubstituted cycloalkyl oraryl, and R¹² and R¹³ optionally together form a substituted orunsubstituted cycloalkyl or aryl; R¹⁴ to R¹⁷ independently are alkyl;and Z is a monatomic or polyatomic ion having a charge sufficient toprovide a neutral compound.

In some embodiments, R³ is a biomolecule-containing group. Thebiomolecule may be an antibody, a peptide, a protein, an amino acid, anucleoside, a nucleotide, a nucleic acid, an oligonucleotide, acarbohydrate, a lipid, a hapten, or a receptor ligand.

In any or all of the above embodiments, R³ may be

where p is 1, 2, 3, 4, or 5, and R^(b) is a biomolecule. In someembodiments, R^(b) is an antibody, a peptide, a protein, an amino acid,a nucleic acid, nucleotide, an oligonucleotide, a lipid, a hapten, or areceptor ligand.

In any or all of the above embodiments, each R^(a) independently may behydrogen or halo, and m is 2 or 3. In any or all of the aboveembodiments, R′⁴ to R′⁷ may be methyl. In any or all of the aboveembodiments, n may be 2.

In any or all of the above embodiments, R⁵, R⁶, R⁸, R¹⁰, R¹¹ and R¹³ maybe hydrogen; R⁴ and R⁹ independently may be lower alkyl or asulfonate-containing group; and R⁷ and R¹² independently may behydrogen, a sulfonate-containing group, or a trialkyl amino group. Insome embodiments, R⁴ and R⁹ are n-propyl or —(CH₂)₄SO₃ ⁻; and R⁷ and R¹²are hydrogen or —SO₃ ⁻Na⁺.

In an independent embodiment, a Cy7 fluorophore has the general formula

where R³, R⁴, R⁷, R⁹, and R¹² are as defined above.

In an independent embodiment, n is 2; R¹ is —CH₂—; m is 2; R² and R¹⁴ toR¹⁷ are methyl; R⁵, R⁶-R⁸ and R¹¹-R¹³ are hydrogen; R⁴ and R⁹ aren-propyl; Z is halide, and R³ is

where X⁻ is a halide.

In an independent embodiment, n is 2; R¹ is —CH₂—; m is 2; R² and R¹⁴ toR¹⁷ are methyl; R⁵, R⁶, R⁸, R¹⁰, R¹¹ and R¹³ are hydrogen; R⁴ and R⁹ are—(CH₂)₄SO₃ ⁻; R⁷ and R¹² are —SO₃ ⁻; Z is an alkali metal cation, and R³is

where M⁺ is a proton or an alkali metal cation.

A method of making the disclosed Cy7 fluorophores includes providing afirst solution comprising a solvent and an ionic precursor of formula(i)

wherein n is 1, 2, or 3, R⁴ to R¹³ independently are H; optionallysubstituted alkyl; optionally substituted amino; or asulfonate-containing group, wherein R⁶ and R⁷ optionally together form asubstituted or unsubstituted cycloalkyl or aryl, and R¹² and R¹³optionally together form a substituted or unsubstituted cycloalkyl oraryl, R¹⁴ to R¹⁷ independently are alkyl, and X is halo.R²(H)N—(R¹)_(m)—OH is added to the first solution, wherein R¹ is —CR^(a)₂— where each R^(a) independently is H, halo, optionally substitutedalkyl, or optionally substituted aryl; m is 2, 3, 4, or 5; and R² isoptionally substituted alkyl, optionally substituted heteroalkyl,optionally substituted aryl, or optionally substituted heteroaryl, andthe first solution is heated at an effective temperature for aneffective period of time to form a solution comprising an ion accordingto formula (ii).

A salt comprising the ion according to formula (ii) is recovered. Asecond solution is formed, wherein the second solution comprises asolvent, the salt, and a compound comprising R³, wherein R³ is amaleimidyl-containing group, a succinimidyl-containing group, optionallysubstituted alkoxy, optionally substituted alkyl carbonyl, optionallysubstituted alkoxy carbonyl, a biomolecule-containing group, or acombination thereof. In some embodiments, the second solution furthercomprises a base. The second solution is reacted under conditionseffective to form an ion according to formula (iii).

A compound according to formula I is recovered, wherein Z is a monatomicor polyatomic ion having a charge sufficient to provide a neutralcompound.

In any or all of the above embodiments, the compound comprising R³ maybe combined withN-[(dimethylamino)-1H-1,2,3-triazolo-[4,5-b]pyridin-1-ylmethylene]-N-methylmethanaminiumhexafluorophosphate N-oxide and diisopropylethylamine before forming thesecond solution.

In any or all of the above embodiments, when R³ terminates in asuccinimidyl moiety, a maleimidyl moiety, —COOH, or —COO⁻, the methodmay further include reacting the compound according to formula I with abiomolecule under effective conditions to form a fluorophore-biomoleculeconjugate.

A method for using embodiments of the disclosed Cy7 fluorophoresincludes contacting a biological sample with a Cy7 fluorophore asdisclosed herein, irradiating the biological sample by application oflight having a wavelength or range of wavelengths in the near-infraredrange, and detecting fluorescence of the irradiated biological sample,wherein fluorescence indicates presence of the Cy7 fluorophore in thebiological sample.

Detecting fluorescence may comprise obtaining a fluorescence-based imageof the irradiated biological sample. In an independent embodiment, theCy7 fluorophore comprises a biomolecule capable of binding to a targetsuspected of being present within the biological sample, fluorescenceindicates the target is present in the biological sample, and the methodfurther includes removing unbound compound from the biological sampleprior to obtaining the image.

In an independent embodiment, the biological sample comprises cells insolution, the Cy7 fluorophore comprises a moiety capable of binding toat least some cells in the solution, and the method further includesperforming flow cytometry to separate cells to which the compound hasbound from cells to which the compound did not bind.

In any or all of the above embodiments, contacting the biological samplewith the Cy7 fluorophore may be performed in vivo by administering thecompound to a subject. In any or all of the above embodiments, detectingfluorescence of the biological sample may be performed ex vivo.

In some embodiments, a Cy7 fluorophore administered in vivo includes abiomolecule capable of binding to a target suspected of being presentwithin the biological sample. In one embodiment, irradiating thebiological sample comprises irradiating a target area of the subjectwith near-infrared radiation, and detecting fluorescence comprisesobtaining an image of the irradiated target area, wherein fluorescencein the image indicates presence of the target in the target area. In anindependent embodiment, the target is an antigen, and the Cy7fluorophore comprises an antibody capable of recognizing and binding tothe antigen. In another independent embodiment, the Cy7 fluorophorecomprises a drug, the biological sample is a bodily fluid or tissue, anddetecting fluorescence of the irradiated biological sample indicatespresence of the drug in the biological sample. In some embodiments, thebiological sample is a tumor and the target area is an area in which thetumor is located. In such embodiments, the biomolecule may be capable ofrecognizing and binding to cells of the tumor, irradiating thebiological sample may comprise irradiating the target area of thesubject with near-infrared radiation, detecting fluorescence mayindicate presence of tumor cells in the target area, and the method mayfurther include excising fluorescent tumor cells from the target area.

The foregoing and other objects, features, and advantages of theinvention will become more apparent from the following detaileddescription, which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1J are absorbance spectra of exemplary C4′-alkyl-etherheptamethine cyanine fluorophores.

FIGS. 2A and 2B are chromatograms of an N-ethanolamine substitutedcompound at 0 minutes (FIG. 2A) and its corresponding C4′-alkyl-etherheptamethine cyanine fluorophore formed via rearrangement after areaction time of 300 minutes (FIG. 2B); the chromatograms were obtainedat 700 nm, and an internal standard was included.

FIGS. 3A and 3B are chromatograms of an N-propanolamine substitutedcompound at 0 minutes (FIG. 3A) and its corresponding C4′-alkyl-etherheptamethine cyanine fluorophore formed via rearrangement after areaction time of 2400 minutes (FIG. 3B); the chromatograms were obtainedat 700 nm, and an internal standard was included.

FIG. 4 is a graph of starting material versus time, showing kineticdifferences in rearrangement of two alkanolamine-substituted compoundsto form exemplary C4′-alkyl-ether heptamethine cyanine fluorophores.

FIGS. 5A and 5B are absorbance and normalized emission spectra of twoexemplary C4′-alkyl-ether heptamethine cyanine fluorophores.

FIG. 6 illustrates the relative stability of an exemplaryC4′-alkyl-ether heptamethine cyanine fluorophore compared to C4′ phenol-and thiol-substituted heptamethine cyanines in the presence of thiolnucleophiles.

FIG. 7 illustrates the reaction between glutathione and a commerciallyavailable heptamethine cyanine fluorophore and shows the formation ofthe glutathione adduct over time.

FIG. 8 illustrates the absence of a reaction between glutathione and anexemplary C4′-alkyl-ether heptamethine cyanine fluorophore.

FIG. 9 is an absorbance spectrum of panitumumab conjugated to anexemplary C4′-alkyl-ether heptamethine cyanine fluorophore.

FIG. 10 is two fluorescence microscopy images of live MDA-MB-468 (HER1+)and MCF-7 (HER1-) cells treated with 100 nM labeled panitumumab andHoechst 33342 (1 μM).

FIG. 11 provides flow cytometry results of MDA-MB-468 and MCF-7 cellstreated with 100 nM labeled panitumumab.

FIGS. 12A-12D are microscopy images of HeLa cells conditioned tooverexpress the folate receptor. FIG. 12A is a fluorescence image of thecells stained with Hoechst 33342.

FIG. 12B is a fluorescence image of the cells incubated with a conjugateof folate and an exemplary C4′-alkyl-ether heptamethine cyaninefluorophore (Folate-Cy7). FIG. 12C is a differential interferencecontrast (DIC) image of the cells. FIG. 12D is a merged image of theimages in FIGS. 12B and 12C.

DETAILED DESCRIPTION

This disclosure concerns a synthetic method for making cyaninefluorophores, particularly stable heptamethine cyanine (Cy7)fluorophores, embodiments of Cy7 fluorophores made by the disclosedmethod, and methods of using the Cy7 fluorophores. The disclosed Cy7fluorophores are C4′-O-alkyl heptamethine cyanines demonstrating opticalproperties that are ideal for near-IR imaging applications and excellentresistance to thiol nucleophiles. Some embodiments of the disclosed Cy7fluorophores are suitable for bioconjugation.

I. Definitions

The following explanations of terms and abbreviations are provided tobetter describe the present disclosure and to guide those of ordinaryskill in the art in the practice of the present disclosure. As usedherein, “comprising” means “including” and the singular forms “a” or“an” or “the” include plural references unless the context clearlydictates otherwise. The term “or” refers to a single element of statedalternative elements or a combination of two or more elements, unlessthe context clearly indicates otherwise.

Unless explained otherwise, all technical and scientific terms usedherein have the same meaning as commonly understood to one of ordinaryskill in the art to which this disclosure belongs. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present disclosure, suitable methods andmaterials are described below. The materials, methods, and examples areillustrative only and not intended to be limiting. Other features of thedisclosure are apparent from the following detailed description and theclaims.

Unless otherwise indicated, all numbers expressing quantities ofcomponents, molecular weights, percentages, temperatures, times, and soforth, as used in the specification or claims are to be understood asbeing modified by the term “about.” Accordingly, unless otherwiseindicated, implicitly or explicitly, the numerical parameters set forthare approximations that may depend on the desired properties soughtand/or limits of detection under standard test conditions/methods. Whendirectly and explicitly distinguishing embodiments from discussed priorart, the embodiment numbers are not approximates unless the word “about”is recited.

Definitions of common terms in chemistry may be found in Richard J.Lewis, Sr. (ed.), Hawley's Condensed Chemical Dictionary, published byJohn Wiley & Sons, Inc., 1997 (ISBN 0-471-29205-2). Definitions ofcommon terms in molecular biology may be found in Benjamin Lewin, GenesVII, published by Oxford University Press, 2000 (ISBN 019879276X);Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, publishedby Blackwell Publishers, 1994 (ISBN 0632021829); and Robert A. Meyers(ed.), Molecular Biology and Biotechnology: a Comprehensive DeskReference, published by Wiley, John & Sons, Inc., 1995 (ISBN0471186341); and other similar references.

In order to facilitate review of the various embodiments of thedisclosure, the following explanations of specific terms are provided:

Alkoxy: A group having the structure —OR, where R is a substituted orunsubstituted alkyl. Methoxy (—OCH₃) is an exemplary alkoxy group. In asubstituted alkoxy, R is alkyl substituted with a non-interferingsubstituent.

Alkoxy carbonyl: A group having the structure —(O)C—O—R, where R is asubstituted or unsubstituted alkyl.

Alkyl: A hydrocarbon group having a saturated carbon chain. The chainmay be branched, unbranched, or cyclic (cycloalkyl). The term loweralkyl means the chain includes 1-10 carbon atoms. Unless otherwisespecified, the term alkyl encompasses substituted and unsubstitutedalkyl.

Alkyl carbonyl: A group having the structure —(O)C—R, where R is asubstituted or unsubstituted alkyl.

Amino: A group having the structure —N(R)R′ where R and R′ areindependently hydrogen, alkyl, heteroalkyl, haloalkyl, aliphatic,heteroaliphatic, aryl (such as optionally substituted phenyl or benzyl),heteroaryl, alkylsulfano, or other functionality. A “primary amino”group is —NH₂. “Mono-substituted amino” means a radical —N(H)Rsubstituted as above and includes, e.g., methylamino,(1-methylethyl)amino, phenylamino, and the like. “Di-substituted amino”means a radical —N(R)R′ substituted as above and includes, e.g.,dimethylamino, methylethylamino, di(1-methylethyl)amino, and the like.The term amino also encompasses charged tri-substituted amino groups,e.g., —N(R)(R′)R″⁺ where R, R′, and R″ are independently hydrogen,alkyl, heteroalkyl, haloalkyl, aliphatic, heteroaliphatic, aryl (such asoptionally substituted phenyl or benzyl), heteroaryl, alkylsulfano, orother functionality.

Aryl: A monovalent aromatic carbocyclic group of, unless specifiedotherwise, from 6 to 15 carbon atoms having a single ring (e.g., phenyl)or multiple condensed rings in which at least one ring is aromatic(e.g., quinoline, indole, benzodioxole, and the like), provided that thepoint of attachment is through an atom of an aromatic portion of thearyl group and the aromatic portion at the point of attachment containsonly carbons in the aromatic ring. If any aromatic ring portion containsa heteroatom, the group is a heteroaryl and not an aryl. Aryl groups aremonocyclic, bicyclic, tricyclic or tetracyclic. Unless otherwisespecified, the term aryl encompasses substituted and unsubstituted aryl.

Bioconjugate: Two or more moieties directly or indirectly coupledtogether, where one of the moieties is a biomolecule. For example, afirst moiety may be covalently or noncovalently (e.g.,electrostatically) coupled to a second moiety. Indirect attachment ispossible, such as by using a “linker” (a molecule or group of atomspositioned between two moieties).

Biomolecule: Any molecule that may be included in a biological system,including but not limited to, a synthetic or naturally occurringantibody, protein (including glycoproteins and lipoproteins), aminoacid, nucleoside, nucleotide, nucleic acid, oligonucleotide, DNA, RNA,carbohydrate (including monosaccharides, disaccharides,oligosaccharides, and polysaccharides), lipid (including fatty acids,monoglycerides, diglycerides, triglycerides, sterols, phospholipids, andfat-soluble vitamins), hapten, a receptor ligand (i.e., a moiety thatbinds to a cellular receptor), and the like.

Cy7: The abbreviation “Cy7” refers to heptamethine cyanine.

Halogen: The terms halogen and halo refer to fluorine, chlorine,bromine, iodine, and radicals thereof.

Heteroalkyl: An alkyl group as defined above containing at least oneheteroatom, such as N, O, S, or S(O)_(n) (where n is 1 or 2). Unlessotherwise specified, the term heteroalkyl encompasses substituted andunsubstituted heteroalkyl.

Heteroaryl: An aromatic compound or group having at least oneheteroatom, i.e., one or more carbon atoms in the ring has been replacedwith an atom having at least one lone pair of electrons, typicallynitrogen, oxygen, phosphorus, silicon, or sulfur. Unless otherwisespecified, the term heteroaryl encompasses substituted and unsubstitutedheteroaryl.

Maleimidyl-containing group: The term maleimidyl-containing groupincludes

groups, where R is substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl.

Near-infrared (near-IR, NIR): Wavelengths within the range of 650-900nm.

Pharmaceutically acceptable carrier: The pharmaceutically acceptablecarriers (vehicles) useful in this disclosure are conventional.Remington: The Science and Practice of Pharmacy, The University of theSciences in Philadelphia, Editor, Lippincott, Williams, & Wilkins,Philadelphia, Pa., 21^(st) Edition (2005), describes compositions andformulations suitable for pharmaceutical delivery of one or moreC4′-alkyl-ether heptamethine cyanine fluorophores as disclosed herein.

In general, the nature of the carrier will depend on the particular modeof administration being employed. For instance, parenteral formulationsusually comprise injectable fluids that include pharmaceutically andphysiologically acceptable fluids such as water, physiological saline,balanced salt solutions, aqueous dextrose, glycerol or the like as avehicle. In some examples, the pharmaceutically acceptable carrier maybe sterile to be suitable for administration to a subject (for example,by parenteral, intramuscular, or subcutaneous injection). In addition tobiologically-neutral carriers, pharmaceutical compositions to beadministered can contain minor amounts of non-toxic auxiliarysubstances, such as wetting or emulsifying agents, preservatives, and pHbuffering agents and the like, for example sodium acetate or sorbitanmonolaurate.

Pharmaceutically acceptable salt: A biologically compatible salt of acompound that can be used as a drug, which salts are derived from avariety of organic and inorganic counter ions well known in the art andinclude, by way of example only, sodium, potassium, calcium, magnesium,ammonium, tetraalkylammonium, and the like; and when the moleculecontains a basic functionality, salts of organic or inorganic acids,such as hydrochloride, hydrobromide, tartrate, mesylate, acetate,maleate, oxalate, and the like. Pharmaceutically acceptable acidaddition salts are those salts that retain the biological effectivenessof the free bases while formed by acid partners that are notbiologically or otherwise undesirable, e.g., inorganic acids such ashydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid, and the like, as well as organic acids such as aceticacid, trifluoroacetic acid, propionic acid, glycolic acid, pyruvic acid,oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid,tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid,methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid,salicylic acid and the like. Pharmaceutically acceptable base additionsalts include those derived from inorganic bases such as sodium,potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper,manganese, aluminum salts and the like. Exemplary salts are theammonium, potassium, sodium, calcium, and magnesium salts. Salts derivedfrom pharmaceutically acceptable organic non-toxic bases include, butare not limited to, salts of primary, secondary, and tertiary amines,substituted amines including naturally occurring substituted amines,cyclic amines and basic ion exchange resins, such as isopropylamine,trimethylamine, diethylamine, triethylamine, tripropylamine,ethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol,dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine,hydrabamine, choline, betaine, ethylenediamine, glucosamine,methylglucamine, theobromine, purines, piperazine, piperidine,N-ethylpiperidine, polyamine resins, and the like. Exemplary organicbases are isopropylamine, diethylamine, ethanolamine, trimethylamine,dicyclohexylamine, choline, and caffeine. (See, for example, S. M.Berge, et al., “Pharmaceutical Salts,” J. Pharm. Sci., 1977; 66:1-19,which is incorporated herein by reference.)

Stokes shift: The difference (in wavelength or frequency units) betweenabsorbance spectrum maximum and the emission spectrum maximum of thesame electronic transition. Typically, the wavelength of maximumfluorescence emission is longer than that of the exciting radiation,i.e., the wavelength of maximum absorbance.

Substituent: An atom or group of atoms that replaces another atom in amolecule as the result of a reaction. The term “substituent” typicallyrefers to an atom or group of atoms that replaces a hydrogen atom, ortwo hydrogen atoms if the substituent is attached via a double bond, ona parent hydrocarbon chain or ring. The term “substituent” may alsocover groups of atoms having multiple points of attachment to themolecule, e.g., the substituent replaces two or more hydrogen atoms on aparent hydrocarbon chain or ring. In such instances, the substituent,unless otherwise specified, may be attached in any spatial orientationto the parent hydrocarbon chain or ring. Exemplary substituents include,for instance, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio,acyl, aldehyde, amido, amino, aminoalkyl, aryl, arylalkyl, arylamino,carbonate, carboxyl, cyano, cycloalkyl, dialkylamino, halo,haloaliphatic (e.g., haloalkyl), haloalkoxy, heteroaliphatic,heteroaryl, heterocycloaliphatic, hydroxyl, isocyano, isothiocyano, oxo,sulfonamide, sulfhydryl, thio, and thioalkoxy groups.

Substituted: A fundamental compound, such as an aryl or aliphaticcompound, or a radical thereof, having coupled thereto one or moresubstituents, each substituent typically replacing a hydrogen atom onthe fundamental compound. Solely by way of example and withoutlimitation, a substituted aryl compound may have an aliphatic groupcoupled to the closed ring of the aryl base, such as with toluene. Againsolely by way of example and without limitation, a long-chainhydrocarbon may have a hydroxyl group bonded thereto.

Succinimidyl-containing group: The term succinimidyl-containing groupincludes

groups, where R is substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl.

Sulfonate-containing group: A group including SO₃ ⁻. The termsulfonate-containing group includes —SO₃ ⁻ and —RSO₃ ⁻ groups, where Ris substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl.

Target: A molecule for which the presence, location and/or concentrationis to be determined. Examples of targets include proteins and nucleicacid sequences present in tissue samples. A target area is an area inwhich a target molecule is located or potentially located.

II. Synthesis of C4′-Alkyl-Ether Heptamethine Cyanines

A synthetic method for making near-IR C4′-alkyl-ether heptamethinecyanine (Cy7) fluorophores involves nitrogen quaternization byelectrophiles to initiate N- to O-transposition in a precursor compound(Scheme 1). With reference to Scheme 1, R is alkyl, heteroalkyl, aryl,or heteroaryl, Y is hydroxyl, E is an electrophile, and B is a base.

An exemplary mechanism of the above process as applied to synthesis ofC4′-alkyl-ether Cy7 fluorophores is shown in Scheme 2, wherein R isoptionally substituted alkyl, optionally substituted heteroalkyl,optionally substituted aryl, or optionally substituted heteroaryl; E isan electrophile; and B is a base.

In general, embodiments of the disclosed C4′-alkyl-ether Cy7fluorophores are synthesized from a halogenated precursor salt. In afirst step (Scheme 3), an ionic halogenated precursor according toformula (i) is reacted with an alkanolamine to produce analkanolamine-substituted precursor according to formula (ii).

With respect to formulas (i) and (ii), n is 1, 2, or 3. R⁴ to R¹³independently are H; optionally substituted alkyl; optionallysubstituted amino; or a sulfonate-containing group, wherein R⁶ and R⁷optionally together form a substituted or unsubstituted cycloalkyl oraryl, and R¹² and R¹³ optionally together form a substituted orunsubstituted cycloalkyl or aryl. R¹⁴ to R¹⁷ independently are alkyl. Xis halo.

R⁴ to R¹³ are selected to provide desired properties of the finalcompound, such as solubility and/or cell permeability. In someembodiments, R⁴, R⁷, R⁹, and R¹² may be selected to provide the desiredcharacteristics. For example, a sulfonate-bearing substituent at one ormore of R⁴, R⁷, R⁹, and R¹² may increase the compound's aqueoussolubility and/or reduce aggregation—desirable traits for biologicalimaging. In an independent embodiment, R⁴ and R⁹ independently are loweralkyl or a sulfonate-containing group. In an independent embodiment, R⁴and R⁹ are the same. In some examples, R⁴ and R⁹ are the same and aren-propyl or n-butylsulfonate (—(CH₂)₄SO₃ ⁻). In an independentembodiment, R⁷ and R¹² independently are hydrogen, asulfonate-containing group, or a trialkyl amino group. In an independentembodiment, R⁷ and R¹² are the same. In some examples, R⁷ and R¹² arethe same and are hydrogen or —SO₃ ⁻ (e.g., —SO₃ ⁻Na⁺). In an independentembodiment, R⁵, R⁶, R⁸, R¹⁰, R¹¹ and R¹³ are hydrogen.

In an independent embodiment, R¹⁴ to R¹⁷ independently are lower alkyl.In some examples, R¹⁴ to R¹⁷ are methyl. In some examples, X is chloro.

A first solution comprising an ionic precursor according to formula (i)is formed and an alkanolamine having the formula R²(H)N—(R¹)_(m)—OH isadded to the first solution. R¹ is —CR^(a) ₂— where each R^(a)independently is H, halogen, optionally substituted alkyl, or optionallysubstituted aryl, and m is 2, 3, 4, or 5. R² is optionally substitutedalkyl, optionally substituted heteroalkyl, optionally substituted aryl,or optionally substituted heteroaryl.

In an independent embodiment, each R^(a) is hydrogen and m is 2 or 3. Insome examples, m is 2. In an independent embodiment, R² is alkyl, suchas lower alkyl. In some examples, R² is methyl. In some examples, thealkanolamine is N-methylethanolamine. The solvent is any solvent ormixture of solvents capable of forming a solution or suspension ofprecursor (i) and the alkanolamine. In some embodiments, the solventcomprises acetonitrile or dimethylformamide.

A reaction between precursor (i) and the alkanolamine proceeds undereffective conditions to form an alkanolamine-substituted precursoraccording to formula (ii). In some examples, the solution is placed in asealed vial and heated at a temperature from 50-80° C. until a change incolor has occurred, indicating that the reaction is complete. Thereaction can be monitored by other means, e.g., liquidchromatography/mass spectroscopy (LC/MS). The solution may be heatedfrom several minutes to several hours, such as from 45 minutes to twohours. Thus, in some embodiments, an effective temperature for thereaction is from 50-80° C., such as from 60-70° C., and an effectiveperiod of time is from 15 minutes to five hours, such as from 45 minutesto two hours. A salt of the alkanolamine-substituted precursor accordingto formula (ii) is recovered from the solution by any suitable meansincluding, but not limited to, precipitation, ion exchange,chromatography (e.g., silica gel chromatography or liquidchromatography, including HPLC), or combinations thereof. In someexamples, alkanolamine-substituted precursors according to formula (ii)exhibit a broad hypsochromic (blue-shifted) absorbance with maxima inthe range of 640-700 nm, characteristic of a C4′-N-linkage.

Subsequently, an electrophile is reacted with thealkanolamine-substituted precursor according to formula (ii) to form aC4′-alkyl-ether heptamethine cyanine ion according to formula (iii)(Scheme 4).

With respect to Scheme 4, R³ is an electrophile capable of initiating anN- to O-rearrangement in the precursor according to formula (ii). Insome embodiments, R³ is a maleimidyl-containing group, asuccinimidyl-containing group, optionally substituted alkoxy, optionallysubstituted alkyl carbonyl, optionally substituted alkoxy carbonyl, abiomolecule-containing group, or a combination thereof. Exemplarymaleimidyl- and succinimidyl-containing groups may further include acarbonyl, alkyl carbonyl, alkoxy, or alkoxy carbonyl group attached tothe ring nitrogen. In some embodiments, R³ is formed by a combination oftwo groups that participate in the rearrangement, e.g., glutaricanhydride and N-hydroxysuccinimide may combine to form thesuccinimidyl-containing group —(O)C(CH₂)₃C(O)O—NC₄H₄O₂.

A solution comprising a solvent, a base, and the salt of the precursoraccording to formula (ii) and a compound comprising R³ is formed.Suitable solvents include solvents in which the salt of precursor (ii)and the compound comprising R³ can be dissolved or suspended.

Exemplary solvents include, but are not limited to, tetrahydrofuran(THF), N,N-dimethylformamide (DMF), dichloromethane (DCM), water, andcombinations thereof.

Suitable bases include inorganic and organic bases. Exemplary basesinclude, but are not limited to, carbonates (e.g., K₂CO₃, Na₂CO₃),hydrogen carbonates (e.g., KHCO₃, NaHCO₃), hydroxides (e.g., KOH, NaOH),and organic amines (e.g., 4-dimethylaminopyridine (DMAP),N,N-diisopropylethylamine (DIPEA),1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC, EDAC, or EDCI), andN-Rdimethylamino)-1H-1,2,3-triazolo-[4,5-b]pyridin-1-ylmethylenel-N-methylmethanaminiumhexafluorophosphate N-oxide (HATU)).

Exemplary compounds comprising R³ include, but are not limited tochloroformates, cyclic anhydrides, alkyl halides, carboxylic acids, andtetrafluoroborates. In some embodiments, the carboxylic acids areactivated carboxylic acids, such as carboxylic acids preactivated withHATU and DIPEA before addition to the solution comprising precursor(ii).

A reaction of precursor (ii) and the compound comprising R³ proceedsunder effective conditions to form a C4′-alkyl-ether heptamethinecyanine ion according to formula (iii). Effective conditions may includereacting at a temperature ranging from room temperature (20-26° C.) to100° C. for a time ranging from a few minutes to several hours. In theexamples disclosed herein, the temperature ranged from room temperatureto 90° C., and the time ranged from 10 minutes to 18 hours. In oneexample, the solution was irradiated with microwave irradiation at 90°C. In various embodiments, the solution is stirred gently, stirredvigorously, or not stirred. The reaction may proceed in a sealed vesselunder an inert atmosphere (e.g., argon, nitrogen). Completion of thereaction may be monitored by any suitable means including, but notlimited to, a visual color change or LC/MS.

A salt comprising the C4′-alkyl-ether heptamethine cyanine ion accordingto formula (iii) is recovered as a C4′-alkyl-ether Cy7 compoundaccording to formula I, wherein Z is a monatomic or polyatomic ionhaving a charge sufficient to provide a neutral compound.

The compound according to formula I is recovered, and optionallypurified, by suitable means. In some embodiments, the compound isrecovered and/or purified by extraction, precipitation, evaporation, ionexchange, chromatography (e.g., silica gel chromatography, HPLC), andcombinations thereof. Some compounds according to formula I exhibit adramatic color change (blue to green) and a bathochromic-shifted(red-shifted) λ_(max) relative to precursor (ii), indicative of theC4′-O-linkage.

Some embodiments of the disclosed compounds are suitable for furtherconjugation to a biomolecule. For example, when R³ terminates in asuccinimidyl moiety, a maleimidyl moiety, —COOH, or —COO⁻, a biomoleculemay be conjugated to the Cy7 fluorophore. Suitable biomolecules include,but are not limited to, antibodies, peptides, amino acids, proteins, andhaptens. One exemplary bioconjugation reaction mediated byN,N′-disuccinimidyl carbonate is shown below in Scheme 5.

III. C4′-Alkyl-Ether Heptamethine Cyanine Fluorophores

Embodiments of C4′-alkyl-ether heptamethine cyanine fluorophoresaccording to general formula I, and pharmaceutically acceptable saltsthereof, are disclosed.

With respect to formula I, m is 2, 3, 4, or 5; n is 1, 2, or 3; IV is—CR^(a) ₂— where each R^(a) independently is H, halo, alkyl, or aryl; R²is optionally substituted alkyl, optionally substituted heteroalkyl,optionally substituted aryl, or optionally substituted heteroaryl; R⁴ toR¹³ independently are H, optionally substituted alkyl, optionallysubstituted amino, or a sulfonate-containing group, wherein R⁶ and R⁷optionally together form a substituted or unsubstituted cycloalkyl oraryl, and R¹² and R¹³ optionally together form a substituted orunsubstituted cycloalkyl or aryl; R¹⁴ to R¹⁷ independently are alkyl;and Z is a monatomic or polyatomic ion having a charge sufficient toprovide a neutral compound. R³ is an electrophile. In some embodiments,R³ is a maleimidyl-containing group, a succinimidyl-containing group,optionally substituted alkoxy, optionally substituted alkyl carbonyl,optionally substituted alkoxy carbonyl, a drug, or abiomolecule-containing group. Exemplary biomolecules include, but arenot limited to, antibodies, peptides, proteins, amino acids,nucleosides, nucleotides, nucleic acids, oligonucleotides,carbohydrates, lipids, haptens, and receptor ligands.

In an independent embodiment, each R^(a) is hydrogen or fluoro and m is2 or 3. In some examples, m is 2. In another independent embodiment, nis 2. In yet another independent embodiment, R² is alkyl, such as loweralkyl. In some examples, R² is methyl. In an independent embodiment, R¹⁴to R¹⁷ independently are lower alkyl. In some examples, R¹⁴ to R¹⁷ aremethyl. In an independent embodiment, R⁴ and R⁹ independently are loweralkyl or a sulfonate-containing group. In another independentembodiment, R⁴ and R⁹ are the same. In some examples, R⁴ and R⁹ are thesame and are n-propyl or n-butylsulfonate (—(CH₂)₄SO₃ ⁻). In anindependent embodiment, R⁷ and R¹² independently are hydrogen, asulfonate-containing group, or a trialkyl amino group. In anotherindependent embodiment, R⁷ and R¹² are the same. In some examples, R⁷and R¹² are the same and are hydrogen or —SO₃ ⁻ (e.g., —SO₃ ⁻Na⁺). In anindependent embodiment, R⁵, R⁶, R⁸, R¹⁰, R¹¹ and R¹³ are hydrogen.

Table 1 includes exemplary compounds according to formula II, in which nis 2, R¹ is —CH₂—, m is 2, R² is methyl, R¹⁴ to R¹⁷ are methyl, and R⁵,R⁶, R⁸, R¹⁰, R¹¹ and R¹³ are hydrogen. Other substituents are asindicated. The maximum absorbance wavelength (λ_(max)) was measured inpH 7.4 phosphate-buffered saline.

TABLE 1 (II)

Cpd Z R³ R⁴ R⁷ R⁹ R¹² 8 I⁻

n-Pr H n-Pr H 9 I⁻

n-Pr H n-Pr H 10 I⁻

n-Pr H n-Pr H 11 I⁻

n-Pr H n-Pr H 12 I⁻

n-Pr H n-Pr H 13 Na⁺

—(CH₂)₄SO₃Na —SO₃Na —(CH₂)₄SO₃Na —SO₃Na 14 Na⁺

—(CH₂)₄SO₃Na —SO₃Na —(CH₂)₄SO₃Na —SO₃Na 15 Na⁺

—(CH₂)₄SO₃Na —SO₃Na —(CH₂)₄SO₃Na —SO₃Na 24 I⁻

n-Pr H n-Pr H 25 Na⁺

—(CH₂)₄SO₃Na —SO₃Na —(CH₂)₄SO₃Na —SO₃Na 26 Na⁺

—(CH₂)₄SO₃Na —SO₃Na —(CH₂)₄SO₃Na —SO₃Na

The disclosed C4′-alkyl-ether Cy7 fluorophores may have a maximumabsorbance wavelength in the range of from 750 to 800 nm, such as from760-775 nm. The fluorophores may have a maximum emission wavelength inthe range of from 775-850 nm, such as from 790-830 nm. The fluorophorestypically have a small Stokes' shift (e.g., ˜25 nm) and high extinctioncoefficients (100,000-250,000 M⁻¹ cm⁻¹).

Some embodiments of the disclosed C4′-alkyl-ether Cy7 fluorophoresexhibit excellent stability, particularly in biological samples that mayinclude thiols (e.g., cysteine, homocysteine, glutathione). Other knownCy7 fluorophores, such as C4′ phenol- and thiol-substituted heptamethinecyanines, which are used widely, rapidly exchange with thiolnucleophiles under aqueous conditions (Zaheer et al., Molecular Imaging2002, 1, 354-360). Problematic consequences have been observed duringconjugation reactions with cysteine-containing peptides andmacromolecules and during DNA sequencing applications, and there is onereport suggesting C4′ exchange reactions can occur intracellularly(Zaheer et al.; Shealy et al., Anal. Chem. 1995, 67, 247-251; Lim etal., JAGS 2014 136, 7018-7025; Pascal et al., J. Phys. Chem. A 2014,118, 4038-4047). In contrast, some embodiments of the disclosedC4′-alkyl-ether Cy7 fluorophores are non-reactive with biological thiols(see Example 2) and exhibit stability (e.g., as determined bysubstantially constant absorbance at λ_(max)) for at least 3 hours, atleast 12 hours, at least one day, or at least 2 days. In one example,compound 13 was stable for at least 3 days with more than 90% of thecompound remaining (i.e., the compound did not react with glutathione).

This disclosure also includes pharmaceutical compositions comprising atleast one C4′-alkyl-ether Cy7 fluorophore. Some embodiments of thepharmaceutical compositions include a pharmaceutically acceptablecarrier and at least one C4′-alkyl-ether Cy7 fluorophore. Usefulpharmaceutically acceptable carriers and excipients are known in theart.

The pharmaceutical compositions comprising one or more C4′-alkyl-etherCy7 fluorophores may be formulated in a variety of ways depending, forexample, on the mode of administration and/or on the location to beimaged. Parenteral formulations may comprise injectable fluids that arepharmaceutically and physiologically acceptable fluid vehicles such aswater, physiological saline, other balanced salt solutions, aqueousdextrose, glycerol or the like. Excipients may include, for example,nonionic solubilizers, such as cremophor, or proteins, such as humanserum albumin or plasma preparations. If desired, the pharmaceuticalcomposition to be administered may also contain non-toxic auxiliarysubstances, such as wetting or emulsifying agents, preservatives, and pHbuffering agents and the like, for example, sodium acetate or sorbitanmonolaurate.

The form of the pharmaceutical composition will be determined by themode of administration chosen. Embodiments of the disclosedpharmaceutical compositions may take a form suitable for virtually anymode of administration, including, for example, topical, ocular, oral,buccal, systemic, nasal, injection, transdermal, rectal, vaginal, etc.,or a form suitable for administration by inhalation or insufflation.Generally, embodiments of the disclosed pharmaceutical compositions willbe administered by injection, systemically, or orally.

Useful injectable preparations include sterile suspensions, solutions oremulsions of the active compound(s) in aqueous or oily vehicles. Thecompositions may also contain formulating agents, such as suspending,stabilizing and/or dispersing agent. The formulations for injection maybe presented in unit dosage form, e.g., in ampules or in multidosecontainers, and may contain added preservatives. The composition maytake such forms as suspension, solutions or emulsions in oily or aqueousvehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents. For example, parenteraladministration may be done by bolus injection or continuous infusion.Alternatively, the fluorophore may be in powder form for reconstitutionwith a suitable vehicle, e.g. sterile water, before use.

Systemic formulations include those designed for administration byinjection, e.g., subcutaneous, intravenous, intramuscular, intrathecalor intraperitoneal injection, as well as those designed for transdermal,transmucosal, oral or pulmonary administration.

Oral formulations may be liquid (e.g., syrups, solutions orsuspensions), or solid (e.g., powder, tablets, or capsules). Oralformulations may be coupled with targeting ligands for crossing theendothelial barrier. Some fluorophore formulations may be dried, e.g.,by spray-drying with a disaccharide, to form fluorophore powders. Solidcompositions prepared by conventional means with pharmaceuticallyacceptable excipients such as binding agents (e.g., pregelatinised maizestarch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers(e.g., lactose, mannitol, microcrystalline cellulose or calcium hydrogenphosphate); lubricants (e.g., magnesium stearate, talc or silica);disintegrants (e.g., potato starch or sodium starch glycolate); orwetting agents (e.g., sodium lauryl sulfate). The tablets may be coatedby methods well known in the art with, for example, sugars, films orenteric coatings. Actual methods of preparing such dosage forms areknown, or will be apparent, to those skilled in the art.

Liquid preparations for oral administration may take the form of, forexample, elixirs, solutions, syrups or suspensions. Such liquidpreparations may be prepared by conventional means with pharmaceuticallyacceptable additives such as suspending agents (e.g., sorbitol syrup,cellulose derivatives or hydrogenated edible fats); emulsifying agents(e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oilyesters, ethyl alcohol, Cremophore™ or fractionated vegetable oils); andpreservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbicacid). The preparations may also contain buffer salts, preservatives,flavoring, coloring and sweetening agents as appropriate. Preparationsfor oral administration may be suitably formulated to give controlledrelease of the fluorophore, as is well known.

For rectal and vaginal routes of administration, the fluorophore(s) maybe formulated as solutions (for retention enemas) suppositories orointments containing conventional suppository bases such as cocoa butteror other glycerides.

For nasal administration or administration by inhalation orinsufflation, the fluorophore(s) can be conveniently delivered in theform of an aerosol spray or mist from pressurized packs or a nebulizerwith the use of a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, fluorocarbons, carbondioxide or other suitable gas. In the case of a pressurized aerosol, thedosage unit may be determined by providing a valve to deliver a meteredamount.

Certain embodiments of the pharmaceutical compositions comprisingfluorophores as described herein may be formulated in unit dosage formsuitable for individual administration of precise dosages. Thepharmaceutical compositions may, if desired, be presented in a pack ordispenser device which may contain one or more unit dosage formscontaining the fluorophore. The pack may, for example, comprise metal orplastic foil, such as a blister pack. The pack or dispenser device maybe accompanied by instructions for administration. The amount offluorophore administered will depend on the subject being treated, thetarget (e.g., the size, location, and characteristics of a tumor), andthe manner of administration, and is known to those skilled in the art.Within these bounds, the formulation to be administered will contain aquantity of the fluorophore disclosed herein in an amount effective toachieve the desired visualization in the subject being treated.

IV. Methods of Use

Embodiments of the disclosed C4′-alkyl-ether Cy7 fluorophores are usefulfor in vitro and in vivo applications where near-IR fluorescence isbeneficial. For example, the fluorophores may be used for in vitro, invivo, or ex vivo imaging, e.g., imaging of drug or antibody conjugates,cell identification, flow cytometry (e.g., fluorescence-activated cellsorting), and super-resolution imaging (e.g., fluorescence resonanceenergy transfer (FRET) imaging). Additionally, the fluorophores may beused in clinical diagnostics and surgery, such as cancer surgery wherenear-IR fluorescence-guided approaches to determine resection marginsare emerging.

A biological sample may be contacted in vivo, ex vivo, or in vitro witha C4′-alkyl-ether Cy7 fluorophore as disclosed herein. For in vivocontact, a pharmaceutical composition comprising the C4′-alkyl-ether Cy7fluorophore may be administered to a subject by any suitable route,e.g., intravenously or orally.

Following contact with the C4′-alkyl-ether Cy7 fluorophore, thebiological sample is irradiated with near-IR radiation, and anyfluorescence of the irradiated biological sample is detected.Irradiation may be performed by application of light having a desiredwavelength or wavelength range within the near-IR range. In someembodiments, suitable light intensities range from 1 mW to 500 mWdepending on the target site and method of application. Near-infraredlight sources can be obtained from commercial sources, includingThorlabs (Newton, N.J.), Laser Components, USA (Hudson, N.H.),ProPhotonix (Salem, N.H.) and others. In some embodiments, irradiationis performed by external application of light to a targeted area. NIRlight is capable of penetrating transcutaneously into tissue to a depthof several centimeters. In other embodiments, irradiation may beperformed by internal application of light, such as by using anendoscope or a fiber optic catheter. Internal application may be usedwhen the target tissue, such as a tumor, is located at a depth that isunsuitable for external light application. For example, an endoscope maybe used for light delivery into the lungs, stomach, or bladder. Thesurface area for light application is generally selected to includetarget tissue, e.g., a tumor or portion of a tumor, or an area of skinexternal to the target tissue. When targeted application of externallight is desired for an in vivo biological sample, the surface area canbe controlled by use of an appropriate light applicator, such as amicro-lens, a Fresnel lens, or a diffuser arrangement. For targetedinternal light application, a desired endoscope or fiber optic catheterdiameter can be selected. In some applications, an indwelling catheterfilled with a light scattering solution may be internally placedproximate the target tissue, and an optical fiber light source may beinserted into the catheter (see, e.g., Madsen et al., Lasers in Surgeryand Medicine 2001, 29, 406-412).

Detection of fluorescence indicates presence of the C4′-alkyl-ether Cy7fluorophore within the biological sample. In some embodiments, detectingfluorescence comprises obtaining a fluorescence-based image of theirradiated biological sample.

In one embodiment, a C4′-alkyl-ether Cy7 fluorophore as disclosed hereincomprises a biomolecule capable of recognizing and binding directly orindirectly, in vitro, in vivo, or ex vivo, to a target (e.g., abiomarker, an antigen, a receptor) present or suspected of being presentin the biological sample. The biological sample is visualized underconditions suitable to produce near-IR fluorescence if theC4′-alkyl-ether Cy7 fluorophore is present in the biological sample. Thepresence of the fluorophore indicates presence of the target. Excessunbound fluorophore may be removed from the biological sample (e.g., bywashing a tissue sample) prior to visualizing the sample to detectfluorescence. In some embodiments, the near-IR fluorescence may bequantified to quantify the amount of target present.

In one non-limiting example, a biological sample (e.g., a bodily fluid,such as urine, blood, saliva, or mucus, or a tissue sample) that maycomprise a target is contacted with a C4′-alkyl-ether Cy7 fluorophoreconjugate comprising an antibody capable of recognizing and binding tothe target. In another non-limiting example, a biological sample thatmay comprise a target is combined with a first antibody capable ofrecognizing and binding to the target; subsequently, the biologicalsample is contacted with a conjugate comprising the C4′-alkyl-ether Cy7fluorophore and an anti-antibody antibody. In another non-limitingexample, the biological sample is contacted with a C4′-alkyl-ether Cy7fluorophore comprising a ligand capable of binding to a receptor. Forinstance, substituent R³ of the C4′-alkyl-ether Cy7 fluorophore mayinclude, or be conjugated to, a receptor ligand capable of binding to areceptor on a cell surface.

In one embodiment, a C4′-alkyl-ether Cy7 fluorophore as disclosed hereincomprises a biomolecule capable of recognizing and binding to a tumorantigen. The biomolecule may be, for example, an antibody thatrecognizes and binds to the tumor antigen. The fluorophore isadministered by a suitable route, e.g., by injection, to a subject witha tumor. Using near-IR fluorescence imaging, the tumor is located andvisualized by its fluorescence. The fluorescence may facilitate completeexcision of the tumor by enabling a user (e.g., a surgeon) to determinewhen the margins are clear, i.e., no fluorescence is detected aftercomplete removal of the tumor. The fluorescence also may be used todetect and monitor tumor growth.

In another embodiment, a C4′-alkyl-ether Cy7 fluorophore as disclosedherein comprises a moiety (e.g., at R³) capable of binding to orassociating with a target molecule in vitro. In one non-limitingexample, the moiety is an oligonucleotide capable of binding to a targetnucleic acid sequence. The nucleic acid sequence may be present, forexample, in a tissue sample (such as formalin-fixed, paraffin-embeddedtissue) or in a gel following gel electrophoresis. The nucleic acidsequence is contacted with the C4′-alkyl-ether Cy7 fluorophore, anyexcess unbound fluorophore is removed, and fluorescence is detected.Fluorescence indicates presence of the target oligonucleotide sequence.In another non-limiting example, the moiety is a receptor ligand capableof binding to a target receptor. The target receptor may be present in atissue sample. The tissue sample is contacted with the C4′-alkyl-etherCy7 fluorophore, any excess unbound fluorophore is removed, andfluorescence is detected. Fluorescence indicates presence of the targetreceptor.

In an independent embodiment, a C4′-alkyl-ether Cy7 fluorophore asdisclosed herein comprises a drug moiety. After administration of theC4′-alkyl-ether Cy7 fluorophore to a subject, near-IR fluorescenceimaging may be used to assess the drug's location within the subjectand/or to monitor drug excretion (e.g., in urine).

The foregoing examples are illustrative only, and other uses arecontemplated. For example, when R³ of the C4′-alkyl-ether Cy7fluorophore comprises a hydroxy or isothiocyanate group, the fluorophoremay be useful as a fluorescent tag for nucleic acids or proteins, e.g.,for use during sequencing, immunoassays, or flow cytometry.

VI. Kits

Kits are also a feature of this disclosure. Embodiments of the kitsinclude at least one compound according to general formula I. In someembodiments, the compound according to general formula I is conjugatedto a biomolecule, e.g., an antibody. In some embodiments, the kits alsoinclude at least one solution in which the compound may be dissolved orsuspended. The kits also may include one or more containers, such as adisposable test tube or cuvette. The kits may further includeinstructions for using the compound and/or for forming a conjugatecomprising the compound according to general formula I and abiomolecule. In some embodiments, the kits further include reagentssuitable for conjugating the compound according to general formula I toa biomolecule.

In some embodiments of the kits, the compound is provided as a solid,and the solution is provided in liquid form. The solution may be asolution suitable for dissolving the compound according to generalformula I so that the dissolved compound may be administered to asubject or so that the dissolved compound may be conjugated to abiomolecule. The solution may be provided at a concentration suitablefor the intended use. Alternatively, the solution may be provided as aconcentrated solution, which is subsequently diluted prior to use. Incertain embodiments, the compound may be premeasured into one or morecontainers (e.g., test tubes or cuvettes).

VI. Examples

General Materials and Methods

Unless stated otherwise, reactions were conducted in oven-driedglassware under an atmosphere of nitrogen or argon using anhydroussolvents (passed through activated alumina columns) All othercommercially obtained reagents were used as received. Thin-layerchromatography (TLC) was conducted with E. Merck silica gel 60 F254pre-coated plates (0.25 mm) and visualized by exposure to UV light (254nm) or stained with anisaldehyde, ceric ammonium molybdate, potassiumpermanganate, or iodine. Flash column chromatography was performed usingnormal phase or reverse phase on a CombiFlash® Rf 200i (Teledyne IscoInc.). Analytical LC/MS was performed using a Shimadzu LCMS-2020 SingleQuadrupole utilizing a Kinetex 2.6 μm C18 100 Å (2.1×50 mm) columnobtained from Phenomenex Inc. Runs employed a gradient of 0→90%MeCN/0.1% aqueous formic acid over 4 minutes at a flow rate of 0.2mL/min. ¹H NMR spectra were recorded on Bruker spectrometers (at 400 or500 MHz) and are reported relative to deuterated solvent signals. Datafor ¹H NMR spectra are reported as follows: chemical shift (6 ppm),multiplicity, coupling constant (Hz), and integration. ¹³C NMR spectrawere recorded on Varian spectrometers (at 100 or 125 MHz). Data for ¹³CNMR spectra are reported in terms of chemical shift. IR spectra wererecorded on a JASCO FT/IR 4100 spectrometer and are reported in terms offrequency of absorption (cm⁻¹). High-resolution LC/MS analyses wereconducted on a Thermo-Fisher LTQ-Orbitrap-XL hybrid mass spectrometersystem with an Ion MAX API electrospray ion source in positive ion mode.Separations were carried out on a narrow-bore (50×2.1 mm), ZorbaxRapid-Resolution, reversed-phase C18 (3.5 μm) column with a flow rate of250 μL/min with a 10 min, 2-90% gradient of MeCN/H₂O containing 0.1%HCOOH. Absorbance traces for quantum yield measurements were performedon a Shimadzu UV-2550 spectrophotometer operated by UVProbe 2.32software. Fluorescence traces and quantum yield measurements wererecorded on a PTI QuantaMaster steady-state spectrofluorimeter operatedby FelixGX 4.0.3 software, with 10 nm excitation and emission slitwidths, 0.1 s integration rate, and enabled emission correction. Dataanalysis and curve fitting were performed using MS Excel 2011 andGraphPad Prism 6. See JOC Standard Abbreviations and Acronyms forabbreviations(http://pubs.acs.org/userimages/ContentEditor/1218717864819/joceah_abbreviations.pdf).

Example 1 Syntheses and Characterization Experimental Procedures

(6): To a solution of IR-780 iodide 3 (100 mg, 0.150 mmol) in methylcyanide (MeCN, 5 mL) was added N-methylethanolamine 7 (60 μL, 0.750mmol). The solution was heated to 70° C. in a sealed vial for 2 hours asthe reaction color transitioned from green to dark blue. After this timeLC/MS analysis showed complete consumption of 3. The reaction mixturewas concentrated in vacuo, and the residue was purified by silica gelchromatography (100% EtOAc, then 0→10% MeOH/DCM) afforded 6 (85 mg, 80%)as a dark blue iridescent solid. Compound 6 had a broad hypsochromicabsorbance with a maximum at 687 nm. ¹H NMR (CDCl₃, 400 MHz) δ 7.69 (d,J=13.0 Hz, 2H), 7.30 (t, J=7.5 Hz, 4H), 7.09 (d, J=7.5 Hz, 2H), 6.89 (d,J=7.9 Hz, 2H), 5.69 (d, J=13.03 Hz, 2H), 4.12-3.94 (m, 4H), 3.81 (t,J=7.4 Hz, 4H), 3.55 (s, 3H), 2.47 (t, J=6.6 Hz, 4H), 1.90-1.76 (m, 6H),1.67 (s, 12H), 1.04 (t, J=7.4 Hz, 6H); ¹³C NMR (CDCl₃, 100 MHz) δ 176.9,168.5, 143.0, 142.2, 140.5, 128.1, 123.5, 123.0, 122.2, 108.7, 95.0,60.0, 59.3, 48.1, 45.3, 44.9, 29.4, 24.9, 21.9, 20.2, 11.7; IR (thinfilm) 1544, 1509, 1444, 1345 cm⁻¹; HRMS (ESI) calculated for C₃₉H₅₂N₃O(M⁺) 578.4110, observed 578.4096.

(7): To a solution of cyanine S1 (115 mg, 0.12 mmol; Lee et al., J. Org.Chem. 2006, 71, 7862-7865) in DMF (2.5 mL) was addedN-methylethanolamine (190 μL, 2.41 mmol). The dark green slurry wassonicated for 5 minutes, then heated to 60° C. in a sealed vial for 45min After this time LC/MS analysis showed complete consumption of S1,and the reaction color had transitioned from green to dark blue. Thereaction was cooled to room temperature and precipitated into Et₂O (100mL) with a 1 mL DMF vial wash. The slurry was centrifuged, thesupernatant discarded, and the blue pellet was resuspended in Et₂O (40mL). The procedure was repeated, and the crude pellet was dissolved in 5mL of water for an ion exchange step. A pipet was filled with 1.5 g ofDowex 50W X8 strongly acidic 200-400 mesh resin, washed with 3 mL ofwater, 5 mL of 1M H₂SO₄, and finally 3 mL of water. The aqueous solutionof the crude 7 was eluted (fast dropwise rate) through the Dowex columninto an aqueous NaHCO₃ solution (200 mg in 2 mL water). After stirringfor 5 minutes, this aqueous solution was purified by reversed-phasechromatography (0→10% MeCN/water) to afford 7 (80 mg, 67%) as a darkblue solid. Compound 7 had a broad hypsochromic absorbance with λ_(max)644 nm (2 μM in PBS, pH 7.4). ¹H NMR (CD₃OD, 400 MHz) δ 7.85-7.77 (m,4H), 7.73 (d, J=13.2 Hz, 2H), 7.16 (d, J=8.3 Hz, 2H), 5.97 (d, J=13.2Hz, 2H), 4.10-3.89 (m, 7H), 3.54 (s, 3H), 2.97-2.81 (m, 4H), 2.57 (t,J=6.6 Hz, 4H), 2.03-1.78 (m, 10H), 1.67 (s, 12H); ¹³C NMR (DMSO-d₆, 100MHz) δ 175.9, 167.6, 143.2, 142.9, 141.0, 139.4, 125.9, 123.4, 119.4,108.5, 95.5, 59.8, 58.6, 50.8, 47.3, 44.2, 42.7, 39.5, 28.7, 25.5, 24.3,22.6, 21.5; IR (thin film) 3412, 1545, 1515, 1478, 1378, 1285 cm⁻¹; HRMS(ESI) calculated for C₄₁H₅₂N₃O₁₃S₄; (M-3H⁻³) 307.4122, observed307.4134.

(8): To a solution of 6 (50 mg, 0.072 mmol; Gorka et al., J. Am. Chem.Soc. dx.doi.org/10.1021/ja5065203) in 1:1/THF:H₂O (1 mL) was addedbenzyl chloroformate (CbzCl, 30 μL, 0.217 mmol) and potassium carbonate(50 mg, 0.362 mmol). The biphasic solution was stirred at roomtemperature for two hours as the reaction color transitioned from darkblue to green. After this time LC/MS analysis showed completeconsumption of 6. The solution was diluted with saturated aqueous sodiumiodide (10 mL), extracted with dichloromethane (2×10 mL), and dried overNa₂SO₄. The solvent removed in vacuo, and the residue was purified bysilica gel chromatography (0→25% MeOH/DCM) affording 8 (54 mg, 88%)) asan iridescent green solid. λ_(max) 763 nm (2 μM in 0.1 M PBS, pH 7.4,with 20% DMSO (v/v), FIG. 1A). ¹H NMR (400 MHz, CD₃CN, 70° C.) δ 8.10(d, J=14.2 Hz, 2H), 7.54-7.14 (m, 13H), 6.09 (d, J=14.2 Hz, 2H), 5.16(s, 2H), 4.15 (t, J=5.7 Hz, 2H), 4.02 (t, J=7.4 Hz, 4H), 3.87 (t, J=5.7Hz, 2H), 3.14 (s, 3H), 2.61 (t, J=6.2 Hz, 4H), 1.92-1.80 (m, 6H), 1.66(s, 12H), 1.03 (t, J=7.4 Hz, 6H). ¹³C NMR (100 MHz, CD₃CN, 70° C.) δ173.6, 171.8, 157.6, 144.1, 142.6, 142.1, 138.7, 129.9, 129.8, 129.2,129.0, 126.1, 124.3, 123.6, 112.2, 100.9, 76.8, 68.3, 50.8, 50.4, 46.9,36.9, 29.0, 25.8, 22.4, 21.8, 11.9. IR (thin film) 1698, 1553, 1505,1361, 1248 cm⁻¹; HRMS (ESI) calculated for C₄₇H₅₈N₃O₃ (M⁺) 712.4473,observed 712.4446. Compound 8 exhibited a bathochromic shifted λ_(max)relative to N-linked 6, a small Stokes shift (λ_(ex)=774 nm, λ_(em)=797nm), and a high absorbance coefficient (ε=187,000 M⁻¹ cm⁻¹).

Table 2 provides 2D-NMR data for compound 8 using the below numberingscheme:

TABLE 2 ¹H (400 MHz), ¹³C (100 MHz), HMBC, and COSY NMR data for 8,CD₃CN, 45° C. atom ¹³C (mult) ¹H mult, J (Hz) HMBC^(a) COSY^(b)  1′100.6 (CH) 6.08 (d, J = 14.2 Hz, 2H) 2,2′,3′  2′  2′ 141.7 (CH) 8.07 (d,J = 14.2 Hz, 2H) 4′,12  1′  3′ 123.9 (C)  4′ 171.3 (C) 11  22.2 (CH₂)1.91-1.78 (m, 2H) 3′,12 12 12  25.5 (CH₂) 2.59 (t, J = 6.1 Hz, 4H)2′,3′,4′,11 11 13  76.6 (CH₂) 4.11 (t, J = 5.5 Hz, 2H) 4′,14 14 14  50.1(CH₂) 3.86 (t, J = 5.5 Hz, 2H) 13,15,16 13 ^(a)Carbons that correlate tothe proton resonance. Optimized for 10 Hz coupling. ^(b)Protons thatcorrelate to the proton resonance.

(9): To a solution of 6 (38 mg, 0.054 mmol) in 1:1/THF:H₂O (1 mL) wasadded 9-fluorenylmethyl chloroformate (FmocCl, 42 mg, 0.16 mmol) andpotassium carbonate (37 mg, 0.27 mmol). The biphasic solution wasstirred vigorously at room temperature for 15 minutes as the reactioncolor transitioned from dark blue to green. After this time LC/MSanalysis showed complete consumption of 6. The solution was diluted withsaturated aqueous sodium iodide (10 mL), extracted with dichloromethane(2×10 mL), and dried over Na₂SO₄. The solvent was removed in vacuo, andthe residue was purified by silica gel chromatography (0→10% MeOH/DCM)affording 9 (48 mg, 95%) as an iridescent green solid. λ_(max) 765 nm (2μM in 0.1 M PBS, pH 7.4, with 20% DMSO (v/v), FIG. 1B). ¹H NMR (400 MHz,CD₃CN, 70° C.) δ 8.02 (d, J=14.2 Hz, 2H), 7.84-7.74 (m, 2H), 7.68-7.58(m, 2H), 7.43-7.19 (m, 12H), 6.08 (d, J=14.2 Hz, 2H), 4.48 (d, J=6.0 Hz,2H), 4.27 (t, J=6.0 Hz, 1H), 4.02 (t, J=7.4 Hz, 4H), 3.98-3.82 (m, 2H),3.77-3.59 (m, 2H), 3.05 (s, 3H), 2.61 (t, J=6.2 Hz, 4H), 1.91-1.79 (m,6H), 1.61 (s, 12H), 1.02 (t, J=7.4 Hz, 5H). IR (thin film) 1699, 1553,1505, 1393, 1362, 1246 cm⁻¹; HRMS (ESI) calculated for C₅₄H₆₂N₃O₃ (M⁺)800.4786, observed 800.4781.

(10): To a solution of 6 (200 mg, 0.28 mmol) in DCM (5 mL) was added4-dimethylaminopyridine (DMPA, 10 mg, 0.084), diisopropylethylamine(iPr₂EtN, 100 μL, 0.56 mmol) and glutaric anhydride (50 mg, 0.43 mmol).The reaction was heated to 35° C. in a sealed vial for 18 hours, duringwhich time the reaction color transitioned from green to dark blue.After this time LC/MS analysis showed complete consumption of 6. Thereaction was diluted with saturated aqueous sodium iodide (10 mL),extracted with dichloromethane (2×10 mL), and dried over Na₂SO₄. Thesolvent was removed in vacuo and the green residue was purified bysilica gel chromatography (0→30% MeOH/DCM) to afford 10 (165 mg, 71%) asan iridescent green solid. λ_(max) 760 nm (2 μM in 0.1 M PBS, pH 7.4,with 0.1% DMSO (v/v), FIG. 1C). ¹H NMR (CD₃CN, 400 MHz, 70° C.) δ 8.12(d, J=14.0 Hz, 2H), 7.51 (d, J=7.4 Hz, 2H), 7.41 (m, 2H), 7.29-7.21 (m,4H), 6.11 (d, J=14.0 Hz, 2H), 4.13 (t, J=6.0 Hz, 2H), 4.03 (t, J=7.4 Hz,4H), 3.92 (t, J=6.0 Hz, 2H), 3.18 (br s, 3H), 2.62 (t, J=6.0 Hz, 4H),2.44-2.36 (m, 2H), 2.34 (t, J=7.3 Hz, 2H), 1.92-1.81 (m, 8H), 1.72 (s,12H), 1.07-1.00 (t, J=7.4 Hz, 6H). IR (thin film) 1723, 1634, 1552,1506, 1366, 1250 cm⁻¹; HRMS (ESI) calculated for C₄₄H₅₈N₃O₄ (M⁺)692.4422, observed 692.4405.

(11): To a solution of 6 (30 mg, 0.043 mmol), acetic acid (AcOH, 5 μL,0.09 mmol), and N,N-diisopropylethylamine (DIPEA, 12 μL, 0.090 mmol) inDCM (2 mL) was added 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide.HCl(EDC.HCl, 16 mg, 0.090 mmol) at room temperature. The reaction washeated to 35° C. in a sealed vial for 18 hours, during which time thereaction color transitioned from dark blue to green. After this timeLC/MS analysis showed complete consumption of 6. The reaction wasdiluted with saturated aqueous sodium iodide (10 mL), extracted withdichloromethane (2×10 mL), and dried over Na₂SO₄. The solvent wasremoved in vacuo and the green residue was purified by silica gelchromatography (0→30% MeOH/DCM) affording 23 mg (72%) of 11 as aniridescent green solid. λ_(max) 760 nm (2 μM in 0.1 M PBS, pH 7.4, with0.1% DMSO (v/v), FIG. 1D). ¹H NMR (400 MHz, CD₃CN, 75° C.) δ 8.11 (d,J=14.2 Hz, 2H), 7.48 (d, J=7.3 Hz, 2H), 7.40 (td, J=7.8, 1.2 Hz, 2H),7.30-7.19 (m, 4H), 6.10 (d, J=14.2 Hz, 2H), 4.19-4.07 (m, 2H), 4.03 (t,J=7.4 Hz, 4H), 3.89 (t, J=5.9 Hz, 2H), 3.24-3.04 (m, 3H), 2.61 (t, J=6.0Hz, 4H), 2.23-2.07 (m, 3H), 1.91-1.80 (m, 6H), 1.71 (br s, 12H), 1.03(t, J=7.4 Hz, 6H); IR (thin film) 1634, 1553, 1505, 1394, 1365, 1248cm⁻¹; HRMS (ESI) calculated for C₄₁H₅₄N₃O₂ (M⁺) 620.4211, observed620.4200.

(12): To a microwave vessel containing 6 (13 mg, 0.019 mmol) indimethylformamide (0.5 mL) was added methyl iodide (MeI, 17 μL, 0.28mmol) and NaHCO₃ (15 mg, 0.28 mmol). The vessel was sealed, purged withargon, and subjected to 90° C. microwave irradiation for 8 hours, duringwhich time the reaction color transitioned from dark blue to green.After this time LC/MS analysis showed complete consumption of 6. Thereaction was precipitated into diethyl ether (10 mL), centrifuged, anddecanted to afford a green residue. The crude material was purified bysilica gel chromatography (0→30% MeOH/DCM) to afford 12 (11 mg, 70%) asa green iridescent solid. λ_(max) 768 nm (2 μM in 0.1 M PBS, pH 7.4,with 0.1% DMSO (v/v), FIG. 1E). ¹H NMR (CD₃OD, 400 MHz) δ 8.02 (d,J=14.0 Hz, 2H), 7.54 (d, J=7.4 Hz, 2H), 7.39 (t, J=7.7 Hz, 2H), 7.31 (d,J=8.0 Hz, 2H), 7.24 (t, J=7.4 Hz, 2H), 6.19 (d, J=14.0 Hz, 2H), 4.59 (t,J=6.2 Hz, 2H), 4.14 (m, 6H), 3.49 (s, 9H), 2.65 (m, 4H), 1.87 (m, 6H),1.76 (s, 12H), 1.04 (t, J=7.4 Hz, 6H); ¹³C NMR (CD₃OD, 100 MHz) δ 173.2,170.2, 143.8, 142.4, 141.0, 129.8, 126.1, 124.0, 123.5, 112.1, 101.3,70.9, 66.4, 55.3, 50.4, 46.6, 29.1, 25.9, 22.1, 21.8, 11.8; IR (thinfilm) 1552, 1503, 1393, 1362, 1247 cm⁻¹; HRMS (ESI) calculated forC₄₁H₅₇N₃O (M⁺²) 303.7245, observed 303.7247.

Table 3 provides 2D-NMR data for compound 12 using the below numberingscheme:

TABLE 3 ¹H (400 MHz), ¹³C (100 MHz), HMBC, and COSY NMR data for 12,CD₃OD atom ¹³C (mult) ¹H mult, J (Hz) HMBC^(a) COSY^(b)  1′ 101.3 (CH)6.20 (d, J = 14.2 Hz, 2H) 2,2′,3,3′  2′  2′ 141.0 (CH) 8.03 (d, J = 14.2Hz, 2H) 2,4′,12  1′  3′ 124.0 (C)  4′ 170.2 (C) 11  22.1 (CH₂) 1.97-1.91(m, 2H) 3′,12 12 12  25.9 (CH₂) 2.75-2.57 (m, 4H) 2′,3′,4′,11 11 13 70.9 (CH₂) 4.60 (t, J = 6.2 Hz, 2H) 4′,14 14 14  66.4 (CH₂) 4.23-4.07(m, 2H) 13,15 13 ^(a)Carbons that correlate to the proton resonance.Optimized for 10 Hz coupling. ^(b)Protons that correlate to the protonresonance.

(13): To a solution of 7 (55 mg, 0.055 mmol) in DMF (0.5 mL) was added4-dimethylaminopyridine (0.4 mg, 0.003), diisopropylethylamine (29 μL,0.17 mmol) and glutaric anhydride (19 mg, 0.17 mmol). The slurry washeated to 35° C. in a sealed vial for 3 hours, during which time thereaction became homogeneous and dark green. After this time LC/MSanalysis showed complete consumption of 7. The reaction was cooled toroom temperature and precipitated into Et₂O (40 mL) with a 1 mL DMF vialwash. The slurry was centrifuged, the supernatant discarded, and thegreen pellet was resuspended in Et₂O (20 mL). The procedure wasrepeated, and the crude was purified by reversed-phase chromatography(0→20% MeCN/0.1% v/v aqueous formic acid). The solvent was evaporated,and the crude material was dissolved in 5 mL of water for an ionexchange step. A pipet was filled with 1.5 g of Dowex 50W X8 stronglyacidic 200-400 mesh resin, washed with 3 mL of water, 5 mL of 1M H₂SO₄,and finally 3 mL of water. The aqueous solution of the crude 13 waseluted (fast dropwise rate) through the Dowex column into an aqueousNaHCO₃ solution (250 mg in 3 mL water). After stirring for 5 minutes,this aqueous solution was purified by reversed-phase chromatography(0→20% MeCN/water) to afford 13 (39 mg, 63%) as a dark green solid.λ_(max) 764 nm (2 μM in 0.1 M PBS, pH 7.4, with 0.1% DMSO (v/v), FIG.1F). ¹H NMR (CD₃OD, 500 MHz, compound exists as a mixture of rotamers;major rotamer is designated by *, minor rotamer denoted by ^(§)) δ 8.18(d, J=14.2 Hz, 1H*), 8.14 (d, J=14.2 Hz, 1H^(§)), 7.96-7.91 (m, 2H*,2H^(§)), 7.89 (d, J=8.4 Hz, 2H*, 2H^(§)), 7.39-7.32 (m, 2H*, 2H^(§)),6.30-6.21 (m, 2H*, 2H^(§)), 4.26-4.10 (m, 6H*, 6H^(§)), 4.04 (t, J=4.5Hz, 1H^(§)), 3.97 (t, J=4.5 Hz, 1H*), 3.28 (s, 3H*), 3.23 (s, 3H^(§)),2.90 (t, J=6.7 Hz, 4H*, 4H^(§)), 2.71-2.64 (m, 4H*, 4H^(§)), 2.58 (t,J=7.7 Hz, 2H^(§)), 2.53 (t, J=7.7 Hz, 2H*), 2.26 (t, J=7.2 Hz, 2H*),2.22 (t, J=7.2 Hz, 2H^(§)), 2.05-1.86 (m, 12H*, 12H^(§)), 1.77 (s,12H*), 1.74 (s, 12H^(§)); IR (thin film) 1723, 1641, 1555, 1503, 1361,1233 cm⁻¹; HRMS (ESI) calculated for C₄₆H₅₈N₃O₁₆S₄; (M-3H⁻³) 345.4228,observed 345.4244.

(14): 6-Maleimidohexanoic acid (12 mg, 0.058 mmol) and HATU(N-Rdimethylamino)-1H-1,2,3-triazolo-[4,5-b]pyridin-1-ylmethylenel-N-methylmethanaminiumhexafluorophosphate N-oxide, 22 mg, 0.058 mmol) were charged to a 1-dramvial. DMF (0.4 mL) and diisopropylethylamine (15 μL, 0.088 mmol) wereadded to the vial under argon, and the resulting homogeneous lightyellow solution was stirred at room temperature for 10 min. Thisactivated ester/DMF solution was then transferred to a vial containing 7(29 mg, 0.029 mmol) and DMF (0.4 mL). The deep blue slurry was heated to35° C. for 1.5 hours, as the reaction color transitioned to green, andafter which LC/MS analysis showed complete consumption of 7. Thereaction was cooled to room temperature and precipitated into Et₂O (40mL) with a 1 mL DMF vial wash. The slurry was centrifuged, thesupernatant discarded, and the green pellet was resuspended in Et₂O (20mL). The procedure was repeated, and the crude material was dissolved in5 mL of water for an ion exchange step. A pipet was filled with 1.5 g ofDowex 50W X8 strongly acidic 200-400 mesh resin, washed with 3 mL ofwater, 5 mL of 1M H₂SO₄, and finally 3 mL of water. The aqueous solutionof the crude 14 was eluted (fast dropwise rate) through the Dowex columninto an aqueous NaHCO₃ solution (100 mg in 2 mL water). After stirringfor 5 minutes, this aqueous solution was purified by reversed-phasechromatography (0→15% MeCN/water) to afford 14 (21 mg, 62%) as a darkgreen solid. λ_(max) 766 nm (2 μM in 0.1 M PBS, pH 7.4, with 0.1% DMSO(v/v), FIG. 1G). ¹H NMR (CD₃OD, 400 MHz, compound exists as a mixture ofrotamers, major rotamer is designated by *, minor rotamer denoted by^(§)) δ 8.18 (d, J=14.1 Hz, 2H*), 8.12 (d, J=14.1 Hz, 2H^(§)), 7.96-7.86(m, 4H*, 4H^(§)), 7.44-7.30 (m, 2H*, 2H^(§)), 6.79 (s, 2H*), 6.76 (s,2H^(§)), 6.32-6.18 (m, 2H*, 2H^(§)), 4.26-4.10 (m, 6H*, 6H^(§)),4.05-3.94 (m, 2H*, 2H^(§)), 3.52 (t, J=6.9 Hz, 2H*), 3.44 (t, J=6.9 Hz,2H^(§)), 3.29 (s, 3H*), 3.18 (s, 3H^(§)), 2.90 (t, J=6.8 Hz, 4H*,4H^(§)), 2.71-2.67 (m, 4H*, 4H^(§)), 2.61 (t, J=7.5 Hz, 2H^(§)), 2.50(t, J=7.4 Hz, 2H*), 2.06-1.88 (m, 10H*, 10H^(§)), 1.77 (s, 12H*), 1.74(s, 12H^(§)), 1.72-1.51 (m, 4H*, 4H^(§)), 1.42-1.26 (m, 2H*, 2H^(§)); IR(thin film) 1701, 1636, 1554, 1507, 1394, 1359, 1254 cm⁻¹; HRMS (ESI)calculated for C₅₁H₆₃N₄O₁₆S₄; (M-3H⁻³) 371.7702, observed 371.7720.

(15): Boc-L-phenylalanine (12 mg, 0.046 mmol) and HATU (17 mg, 0.046mmol) were charged to a 1-dram vial. DMF (0.4 mL) anddiisopropylethylamine (12 μL, 0.069 mmol) were added to the vial underargon, and the resulting homogeneous light yellow solution was stirredat room temperature for 10 min. This activated ester/DMF solution wasthen transferred to a vial containing 7 (23 mg, 0.023 mmol) and DMF (0.4mL). The deep blue slurry was heated to 35° C. for 1.5 hours as thereaction color transitioned to green, at which point LC/MS analysisshowed complete consumption of 7. The reaction was cooled to roomtemperature and precipitated into Et₂O (40 mL) with a 1 mL DMF vialwash. The slurry was centrifuged, the supernatant discarded, and theblue pellet was resuspended in Et₂O (20 mL). The procedure was repeated,and the crude material was dissolved in 5 mL of water for an ionexchange step. A pipet was filled with 1.5 g of Dowex 50W X8 stronglyacidic 200-400 mesh resin, washed with 3 mL of water, 5 mL of 1M H₂SO₄,and finally 3 mL of water. The aqueous solution of the crude 15 waseluted (fast dropwise rate) through the Dowex column into an aqueousNaHCO₃ solution (80 mg in 2 mL water). After stirring for 5 minutes,this aqueous solution was purified by reversed-phase chromatography(0→20% MeCN/water) to afford 15 (21 mg, 73%) as a dark green solid.λ_(max) 768 nm (2 μM in 0.1 M PBS, pH 7.4, with 0.1% DMSO (v/v), FIG.1H). ¹H NMR (CD₃OD, 500 MHz, compound exists as a mixture of rotamers,major rotamer is designated by *, minor rotamer denoted by ^(§)) δ 8.16(d, J=14.0 Hz, 2H^(§)), 8.09 (d, J=14.0 Hz, 2H*), 7.94-7.83 (m, 4H*,4H^(§)), 7.37 (d, J=8.3 Hz, 2H*, 2H^(§)), 7.32-7.09 (m, 4H*, 4H^(§)),6.31-6.20 (m, 2H*, 2H^(§)), 5.00-4.95 (m, 1H*), 4.82-4.78 (m, 1H^(§)),4.24-4.14 (m, 4H*, 4H^(§)), 4.15-3.60 (m, 4H*, 4H^(§)), 3.15 (s, 3H*),3.03 (s, 3H^(§)), 3.00-2.85 (m, 6H*, 6H^(§)), 2.76-2.62 (m, 4H*,4H^(§)), 2.06-1.87 (m, 10H*, 10H^(§)), 1.84-1.32 (m, 21H*, 21H^(§)); IR(thin film) 1701, 1643, 1555, 1507, 1394, 1361, 1255 cm⁻¹; HRMS (ESI)calculated for C₅₅H₆₉N₃O₁₆S₄; (M-3H⁻³) 389.7858, observed 389.7875.

(16): To a mixture of MeO₃ ⁺BF₄ ⁻ (38 mg, 0.26 mmol) and1,8-bis(dimethyl-amino)naphthalene (Proton Sponge®, Sigma-Aldrich, 16mg, 0.052 mmol) in DCM (0.5 mL) under argon was added a solution of 6(36 mg, 0.052 mmol) in DCM (2 mL) at room temperature. The blue reactionwas heated to 35° C. for 2 hours, at which time LC/MS analysis showedcomplete consumption of 6. The reaction was quenched with saturatedaqueous NaHCO₃ (10 mL) and extracted with dichloromethane (15 mL). Theorganic layer was washed with saturated aqueous sodium iodide (2×5 mL)and dried over Na₂SO₄. The solvent was removed in vacuo and the blueresidue was purified by silica gel chromatography (0→10% MeOH/DCM)affording 31 mg (85%) of 16 as an iridescent blue solid. λ_(max) 668 nm(2 μM in 0.1 M PBS, pH 7.4, with 0.1% DMSO (v/v), FIG. 1I). ¹H NMR (500MHz, CDCl₃) δ 7.60 (d, J=13.4 Hz, 2H), 7.34-7.27 (m, 4H), 7.10 (t, J=7.5Hz, 2H), 6.95 (d, J=7.9 Hz, 2H), 5.76 (d, J=13.4 Hz, 2H), 3.96 (t, J=4.9Hz, 2H), 3.86 (t, J=7.3 Hz, 4H), 3.78 (t, J=5.0 Hz, 2H), 3.48 (s, 3H),3.39 (s, 3H), 2.46 (t, J=6.5 Hz, 4H), 1.91-1.78 (m, 6H), 1.64 (s, 12H),1.04 (t, J=7.4 Hz, 6H); ¹³C NMR (125 MHz, CDCl₃) δ 175.7, 168.7, 142.9,142.2, 140.3, 128.3, 123.6, 123.3, 122.1, 109.1, 95.5, 70.1, 58.7, 57.2,48.0, 45.0, 45.0, 29.1, 24.7, 21.8, 20.3, 11.7. IR (thin film) 1547,1506, 1449, 1343, 1252 cm⁻¹; HRMS (ESI) calculated for C₄₀H₅₄N₃O (M⁺)592.4261, observed 592.4246.

This example demonstrated that, consistent with a requirement forinitial N-activation, different methylation conditions produceddivergent reaction pathways. While methyl iodide yielded the desiredrearrangement product (compound 12), trimethyloxonium tetrafluoroborateafforded instead the methyl ether 16 without the desired rearrangement.

(17): To a mixture of 6 (30 mg, 0.043 mmol) and 2,6-lutidine (20 μL,0.17 mmol) in DCM (1 mL) was added acetyl chloride (9.0 μL, 0.13 mmol).The blue solution was stirred for 16 hours at room temperature, at whichtime LC/MS analysis showed complete consumption of 6. The solution wasdiluted with saturated aqueous sodium iodide (5 mL), extracted withdichloromethane (2×10 mL), and the organic layer dried over Na₂SO₄. Thesolvent was removed in vacuo, and the blue residue was purified bysilica gel chromatography (0→10% MeOH/DCM) affording 28 mg (87%) of 17as an iridescent blue solid. λ_(max) 689 nm (2 μM in 0.1 M PBS, pH 7.4,with 0.1% DMSO (v/v), FIG. 1J). ¹H NMR (400 MHz, CD₃CN) δ 7.62 (d,J=13.6 Hz, 2H), 7.46-7.38 (d, J=7.3 Hz, 2H), 7.33 (td, J=7.7, 1.2 Hz,2H), 7.19-7.07 (m, 4H), 5.92 (d, J=13.6 Hz, 2H), 4.31 (t, J=5.0 Hz, 2H),3.99-3.82 (m, 6H), 3.40 (s, 3H), 2.48 (t, J=6.3 Hz, 4H), 2.13 (br s,3H), 1.86-1.70 (m, 6H), 1.63 (s, 12H), 1.00 (t, J=7.4 Hz, 6H); ¹³C NMR(100 MHz, CD₃CN) δ 175.3, 171.4, 170.7, 144.1, 143.4, 141.6, 129.3,125.0, 124.4, 123.1, 110.8, 97.3, 63.5, 57.6, 49.1, 45.9, 45.7, 29.3,25.5, 22.8, 21.2, 21.0, 11.7. IR (thin film) 1735, 1543, 1503, 1341,1249 cm⁻¹; HRMS (ESI) calculated for C₄₁H₅₄N₃O₂ (M⁺) 620.4211, observed620.4185.

This example demonstrated that, consistent with a requirement forinitial N-activation, different acylation conditions produced divergentreaction pathways. Unlike the peptide coupling conditions used toproduce compound 11, a combination of acetyl chloride and a weak base,2,6-lutidine, provided only O-acylation product 17 in high yield.

(18): To a solution of IR-780 iodide 3 (315 mg, 0.472 mmol) in MeCN (3mL) was added 3-methylamino-1-propanol (230 μL, 2.36 mmol). The solutionwas heated to 80° C. in a sealed vial for 10 minutes as the reactioncolor transitioned from green to dark blue. After this time LC/MSanalysis showed complete consumption of 3. The reaction mixture wasconcentrated in vacuo, and the residue was purified by silica gelchromatography (0→10% MeOH/DCM) to afford 18 (285 mg, 84%) as a darkblue iridescent solid. ¹H NMR (CD₃CN, 400 MHz) δ 7.51 (d, J=13.3 Hz,2H), 7.38 (d, J=7.3 Hz, 2H), 7.33-7.25 (m, 2H), 7.13-7.03 (m, 4H), 5.83(d, J=13.4 Hz, 2H), 3.86 (m, 6H), 3.55 (q, J=5.6 Hz, 2H), 3.40 (s, 3H),3.02 (t, J=4.9 Hz, 1H), 2.47 (t, J=6.6 Hz, 4H), 2.01-1.93 (m, 2H),1.83-1.69 (m, 6H), 1.59 (s, 12H), 0.98 (t, J=7.4 Hz, 6H); ¹³C NMR(CD₃CN, 125 MHz) δ 176.8, 169.4, 144.2, 142.2, 141.3, 129.1, 124.3,123.8, 122.9, 110.3, 95.9, 59.6, 56.8, 48.6, 45.7, 45.4, 32.5, 29.4,25.4, 22.6, 20.8, 11.6; IR (thin film) 3339, 1540, 1508, 1449, 1344,1256 cm⁻¹; HRMS (ESI) calculated for C₄₀H₅₄N₃O (M⁺) 592.4261, observed592.4255.

(19): To a solution of alcohol 18 (25 mg, 0.035 mmol), acetic acid (8μL, 0.14 mmol), and DIPEA (6 μL, 0.035 mmol) in DCM (2 mL) was addedEDC.HCl (27 mg, 0.14 mmol) at room temperature. The vial was sealed andthe reaction was heated to 40° C. for 48 hours. After this time LC/MSanalysis showed complete consumption of 18. The reaction was dilutedwith saturated aqueous sodium iodide (10 mL), extracted withdichloromethane (2×10 mL), and dried over Na₂SO₄. The solvent removed invacuo, and the green residue was purified by silica gel chromatography(0→10% MeOH/DCM) affording 19 mg (72%) of 19 as an iridescent greensolid. ¹H NMR (400 MHz, CD₃CN, 72° C.; compound exists as a mixture ofrotamers, major rotamer is designated by *, minor rotamer denoted by §)δ 8.11 (d, J=14.2 Hz, 2H), 7.49-7.45 (m, 2H), 7.42-7.37 (m, 2H),7.27-7.20 (m, 4H), 6.10 (d, J=14.2 Hz, 2H), 4.13-3.98 (m, 6H), 3.68-3.51(m, 2H), 3.04 (br s, 3H*), 2.94 (br s, 3H^(§)), 2.61 (t, J=6.2 Hz, 4H),2.31-2.17 (m, 2H), 2.07 (br s, 3H^(§)), 2.00 (br s, 3H*), 1.91-1.80 (m,6H), 1.70 (s, 12H), 1.03 (t, J=7.4 Hz, 6H). IR (thin film) 1632, 1551,1505, 1393, 1360, 1245 cm⁻¹; HRMS (ESI) calculated for C₄₂H₅₆N₃O₂ (M⁺)634.4367, observed 634.4355.

(21): 21 was prepared according to procedure of Strekowski et. al. (J.Org. Chem. 1992, 57, 4578-4580). To a solution of S1 (38 mg, 0.040 mmol)in DMF (0.7 mL) and MeOH (0.7 mL) was added phenol (26 mg, 0.28 mmol)and diisopropylethylamine (48 μL, 0.28 mmol). The dark green solutionwas heated to 45° C. in a sealed vial for 3 hours, after which timeLC/MS analysis showed complete consumption of S1. The reaction wascooled to room temperature and precipitated into Et₂O (40 mL). Theslurry was centrifuged, the supernatant discarded, and the green pelletwas resuspended in Et₂O (20 mL). The procedure was repeated, and thecrude material was dissolved in 5 mL of water for an ion exchange step.A pipet was filled with 1.5 g of Dowex 50W X8 strongly acidic 200-400mesh resin, washed with 3 mL of water, 5 mL of 1M H₂SO₄, and finally 3mL of water. The aqueous solution of the crude 21 was eluted (fastdropwise rate) through the Dowex column into an aqueous NaHCO₃ solution(70 mg in 2 mL water). After stirring for 5 minutes, this aqueoussolution was loaded directly onto a C₁₈Aq column and purified byreversed-phase chromatography (0→25% MeCN/water) to afford 21 (32 mg,80%) as a dark green solid. ¹H NMR (CD₃OD, 400 MHz) δ 8.02 (d, J=14.2Hz, 2H), 7.85 (dd, J=8.3, 1.7 Hz, 2H), 7.78 (d, J=1.7 Hz, 2H), 7.47-7.37(m, 2H), 7.34 (d, J=8.3 Hz, 2H), 7.14 (d, J=8.7 Hz, 2H), 7.06 (t, J=7.6Hz, 1H), 6.26 (d, J=14.2 Hz, 2H), 4.28-4.05 (m, 4H), 2.89 (t, J=6.8 Hz,4H), 2.82-2.75 (m, 4H), 2.11-2.01 (m, 2H), 2.01-1.87 (m, 8H), 1.37 (s,12H); ¹³C NMR (CD₃OD, 100 MHz) δ 174.0, 165.9, 161.3, 144.9, 143.8,143.1, 142.3, 131.5, 128.1, 124.6, 123.7, 121.2, 115.8, 111.6, 101.9,51.7, 50.2, 45.0, 28.1, 27.1, 25.2, 23.5, 22.4; IR (thin film) 1558,1512, 1431, 1400, 1361 cm⁻¹; HRMS (ESI) calculated for C₄₄H₄₉N₂O₁₃S₄;(M-3H⁻³) 313.7367, observed 313.7361.

(22): 22 was prepared according to procedure of Strekowski et. al. (J.Org. Chem. 1992, 57, 4578-4580). To a solution of S1 (35 mg, 0.037 mmol)in DMF (2 mL) and MeOH (50 μL) was added β-mercaptoethanol (10 μL, 0.15mmol) and diisopropylethylamine (26 μL, 0.15 mmol). The dark greensolution was heated to 35° C. in a sealed vial for 4 hours. The reactionwas cooled to room temperature and precipitated into Et₂O (40 mL). Theslurry was centrifuged, the supernatant discarded, and the green pelletwas resuspended in Et₂O (20 mL). The procedure was repeated, and thecrude material was dissolved in 5 mL of water for an ion exchange step.A pipet was filled with 1.5 g of Dowex 50W X8 strongly acidic 200-400mesh resin, washed with 3 mL of water, 5 mL of 1M H₂SO₄, and finally 3mL of water. The aqueous solution of the crude 22 was eluted (fastdropwise rate) through the Dowex column into an aqueous NaHCO₃ solution(70 mg in 2 mL water). After stirring for 5 minutes, this aqueoussolution was loaded directly onto a C₁₈Aq column and purified byreversed-phase chromatography (0→20% MeCN/water) to afford 22 (23 mg,64%) as a dark green solid. ¹H NMR (DMSO-d₆, 500 MHz) δ 8.77 (d, J=14.1Hz, 2H), 7.77 (d, J=1.6 Hz, 2H), 7.65 (dd, J=8.3, 1.6 Hz, 2H), 7.38 (d,J=8.3 Hz, 2H), 6.36 (d, J=14.1 Hz, 2H), 4.96 (t, J=5.3 Hz, 1H),4.28-4.11 (m, 4H), 3.50 (q, J=6.3 Hz, 2H), 2.87 (t, J=6.7 Hz, 2H),2.71-2.61 (m, 4H), 2.56-2.51 (m, 4H), 1.89-1.72 (m, 10H), 1.70 (s, 12H);¹³C NMR (DMSO-d₆, 125 MHz) δ 171.9, 155.2, 145.0, 145.0, 142.2, 140.3,133.4, 126.2, 119.8, 110.4, 101.7, 60.1, 50.7, 48.8, 43.7, 39.1(observed by HSQC correlation), 27.4, 26.0, 25.7, 22.5, 20.7; IR (thinfilm) 3410, 1530, 1507, 1432, 1348, 1248 cm⁻¹; HRMS (ESI) calculated forC₄₀H₄₉N₂O₁₃S₅; (M-3H⁻³) 308.3941, observed 308.3937.

Example 2

Kinetic Effect of Alkanolamine Chain Length on Rearrangement

The rearrangement kinetics of the N-ethanolamine substituted compound 6and its N-propanolamine homolog 18 were compared.

To a solution of alcohol 6 or 18 (0.015 mmol), acetic acid (0.060 mmol),and DIPEA (0.015 mmol) in DCM (0.5 mL) was added an internal standard(IR-780, 0.015 mmol). A t=0 min, a sample was withdrawn and analyzed byHPLC. EDC.HCl (0.060 mmol) was added and the reaction was heated to 35°C. At the given time points in FIG. 2 (compound 6 to 11) and FIG. 3(compound 18 to 19), a 5 μL aliquot was withdrawn and diluted in 500 μLmethanol (to halt the reaction progress). The sample was then analyzedon an Agilent 1260 Infinity HPLC utilizing a Kinetex 5 μm C6-Phenyl 110Å (4.6×250 mm) column (Phenomenex Inc.) with a gradient of 5→98% (10min) to 98→5% (1 min) MeCN/0.1% aqueous trifluoroacetic acid at a flowrate of 2.0 mL/min. Experiments were run in duplicate and plotted witherror bars derived from the standard deviation (<5% in all cases). Thepeaks were assigned by comparing retention times with purifiedstandards.

While 6 reacted relatively rapidly (t_(1/2)=37 min) to form 11, 18proceeded much more slowly (t_(1/2)=720 min), though ultimately in goodconversion to 19, and with satisfactory isolated yield (72%) (FIG. 4).The kinetic dependence on ring size (5-membered faster than 6-membered)suggested that acyl-ammonium formation, which is likely reversible,precedes rate determining, and presumably, irreversible tetrahedralintermediate formation.

Without wishing to be bound by a particular theory of operation, it isthought that the rearrangement proceeds through tetrahedralintermediates. However, concerted displacement orhemolysis/recombination pathways cannot be completely excluded.

Example 3 Reversibility of the Rearrangement

The reversibility of the rearrangement was examined

When Fmoc-protected 9 was exposed to piperidine in DMF, theC4′-N-methylethanolamine-substituted product 6, its synthetic precursor,was rapidly generated. To a solution of 9 (18 mg, 0.019 mmol) in DMF(300 μL) was added piperidine (10 μL, 0.097 mmol). The green solutionwas stirred at room temperature for 20 minutes, during which time thereaction color transitioned to dark blue and a precipitate resulted. Atthis time LC/MS analysis showed a complete conversion of 9 to 6. Thereaction mixture was diluted with DCM (20 mL), washed with water (4×5mL), saturated aqueous sodium iodide (2×5 mL), and dried over Na₂SO₄.The volatiles were concentrated in vacuo, and the blue residue waspurified by silica gel chromatography (50% EtOAc/DCM, 0→25% MeOH/DCM)affording 10 mg (73%) of product. The analytical data were consistentwith those for compound 6.

Without wishing to be bound by any particular theory of operation, thisprocess likely occurred through the intermediacy of O-linked species 20.This observation suggests a thermodynamic preference for C4′-N-linkage.The facile conversion of 9 to 6 altered the absorbance maxima by almost90 nm, suggesting potential for various optical sensing applications.

Example 4 Determination of Quantum Yields and Molar AbsorptionCoefficients

Quantum yields (Φ_(f)) were determined in methanol relative to ICG andIR783 (Φf=0.078 and 0.084, respectively; James et al., Theranostics2013, 3, 692-702), from plots of integrated fluorescence intensity vs.absorbance, according to the following relationship:

$\Phi_{x} = {{\Phi_{st}\left( \frac{{Grad}_{x}}{{Grad}_{st}} \right)}\left( \frac{\eta_{x}}{\eta_{st}} \right)}$where subscripts st and x denote standard and test sample, respectively,0 is the fluorescence quantum yield, Grad is the gradient of theintegrated fluorescence intensity vs. absorbance plot, and η is therefractive index of the solvent (Parker, et al., Analyst 1960, 85, 587;Williams, et al., Analyst. 1983, 108, 1067; Rurack, et al., Anal. Chem.2011, 83, 1232; Samanta, et al., Chem. Commun. 2010, 46, 7406).Measurements were performed in 10 mm path length quartz cuvettes (Hellma111-QS), maintained at 25° C., with the absorbance of all dye solutions≤0.08 in order to maximize illumination homogeneity and opticaltransparency and minimize reabsorption effects (Samanta, et al., Chem.Commun. 2010, 46, 7406). ICG and IR783 standards and test dye solutionswere excited at 70 nm below their absorbance maxima.

Molar absorption coefficients (ε) were determined in PBS using Beer'slaw, from plots of absorbance vs. concentration. Measurements wereperformed in 10 mm path length quartz cuvettes (Hellma 111-QS),maintained at 25° C., with absorbance at the highest concentration ≤0.08(see above).

Representative compounds 8 (5 μM in methanol) and 13 (3 μM in methanol)were found to possess similar absorbance and normalized emission spectra(FIGS. 5A and 5B, respectively) and improved quantum yield relative to astandard heptamethine cyanine, indocyanine green (ICG) (Table 4).

TABLE 4 Compound λ_(max) (nm) λ_(em) (nm) ε (M⁻¹cm⁻¹) Φ_(f) ICG 785 822204,000 0.078  8^(a) 774 797 187,000 0.22 13^(a) 774 798 214,000 0.23^(a)Measured in methanol relative to ICG.

Example 5 Stability of C4′-Alkyl-Ether Heptamethine Cyanine Fluorophores

The thiol reactivity of 13 was compared with phenol-substituted 21 andS-mercaptoethanol-substituted 22.

Stock solutions (5 mM) of 13, 21, and 22 were prepared in DMSO. A fivehundred-fold dilution in 50 mM PBS buffer (pH=7.4) was performed toyield 10 μM samples. The samples were analyzed by HPLC (t=0 min) and 5μL of a 0.2 M glutathione solution in deionized water was added toafford a 1 mM final glutathione concentration. The samples werecontinuously analyzed every 20 minutes by HPLC, and the integrated peakareas of absorbance at 780 nm from the starting dyes were plotted versustime. The samples were analyzed on an Agilent 1260 Infinity HPLCutilizing a Kinetex 5 μm Biphenyl 100 Å (4.6×250 mm) column (PhenomenexInc.) with a gradient of 2→98% (4.7 min) to 98→2% (1 min) MeCN/10 mMammonium carbonate at a flow rate of 1.5 mL/min Experiments were run induplicate and plotted with error bars derived from the standarddeviation (<5% in all cases). After the reaction of 21 and GSH achievedfull conversion the reaction was directly purified by semi-prep HPLC toobtain glutathione-cyanine adduct 23, which was analyzed by HRMS. HRMS(ESI) calculated for C₄₈H₆₀N₅O₁₈S₅; (M-3H⁻³) 384.7507, observed384.7500.

With compounds 21 and 22, rapid conversion to the glutathione adduct 23was observed (t_(1/2)=95 and 40 minutes, respectively), as shown in FIG.6. By contrast, the alkyl-ether 13 showed no decomposition over the sametime period and >90% was present after 3 days.

Without wishing to be bound by any particular theory of operation, thedramatic differences in reactivity are thought to stem from thesignificantly different energetic requirements involved in ionizingphenolate, thiolate, and alkoxide leaving groups. This study clearlydemonstrates the superior chemical stability of these new C4′-alkylethers over conventional phenol or thiol-substituted heptamethinecyanines.

Solutions of commercially available IRDye 800 CW (a phenol-substitutedheptamethine cyanine available from Li-Cor Biotechnology, Lincoln,Nebr.) and compound 13 (Glut-Cy-503), 10 μM, were prepared in pH 7.4phosphate-buffered saline and exposed to physiologically relevantconcentrations of glutathione (GSH, 1 mM) at room temperature. Whereasthe phenol-based fluorophore degraded within 3 hours to provide theglutathione (—S—R) adduct (t_(1/2)=50 minutes, FIG. 7, measured by HPLCarea under curve), the C4′-alkyl-ether Cy7 compound 13 showed minimaldecomposition over the same time period and was stable for >2 days (FIG.8). Upon analysis by LCMS, signals consistent with the glutathioneadduct were observed concomitant with the disappearance of the phenol(IRDye 800 CW) (FIG. 7). In contrast, no formation of the glutathioneadduct was observed with compound 13.

Example 6 Antibody Labeling

NHS ester synthesis: A suspension of 13 (14 mg, 0.012 mmol) in toluene(5 mL) was concentrated in vacuo to azeotropically remove water. Asolution of TSTU (N,N,N′,N′-tetramethyl-O—(N-succinimidyl)uraniumtetrafluoroborate, 7.5 mg, 0.025 mmol) and diisopropylethylamine (2.2μL, 0.012 mmol, 1 equivalent) in DMF (500 μL) was added to 13 under anargon atmosphere. This solution was heated to 35° C. for 16 hours. Afterthis time LC/MS analysis showed complete consumption of 13. The reactionwas subsequently precipitated into ethyl acetate (45 mL), centrifuged,and the supernatant decanted off. The green pellet was resuspended inEt₂O (20 mL), and the centrifugation procedure was repeated twice. Thecrude solid (S2) was placed under vacuum (<0.5 Torr) for 1 hour and useddirectly in the antibody conjugation step.

Panitumumab conjugation (Bhattacharyya et al., Med Chem. Comm. 2014, 5,1337-1346): To 500 μL of 1M PBS (pH 8.5) in a 1.5 mL microcentrifugetube was added 500 μL of panitumumab solution, a 5 mg/mL solutionacquired from Amgen, and 38 μL of a 5 mM DMSO solution of S2 undersubdued lighting. The microcentrifuge tube was gently inverted twice tomix the solution and placed in a dark box on a slowly rocking platformfor one hour at room temperature. A G10 Sephadex column acquired from GEHealthcare was primed with six column volumes of 0.9% saline. After onehour on the rocking platform, the solution was eluted through the columnwith 0.9% saline and collected in a 2 mL Cryotube. 1 mL was collectedper fraction. The dye and antibody concentrations were determined byUV-Vis spectroscopy using Beer-Lambert's Law C=A/εl with the extinctioncoefficient of 168,000 M⁻¹ cm⁻¹ at 774 nm in 1M PBS and 200,000 M⁻¹ cm⁻¹at 280 nm 1:1 PBS:MeOH for 13 and panitumumab, respectively. The degreeof labeling (DOL), the average number of dye molecules (compound 13)bonded to one antibody, was determined as the quotient of dyeconcentration and antibody concentration. A DOL of 2.1-2.2 was obtained.The solution of antibody-conjugate was then filtered through a 0.22 μmsterile filter acquired from Acrodisc for storage at 4° C. A UV-Vis-NIRabsorbance spectrum of the panitumumab-13 conjugate is shown in FIG. 9.

Cell culture: DA-MB-468 (EGFR/HER1 overexpression; Rae et al., BreastCancer Res. Treat 2004, 87, 87-95; Mamot et al., Cancer Res. 2003, 63,3154-3161) and MCF7 (normal EGFR/HER1 expression; Williams et al.,Analyst 1983, 108, 1067-1071) human breast cancer cell lines wereobtained from the National Cancer Institute Developmental TherapeuticsProgram, DCTD Tumor Repository. MDA-MB-468 was cultured in RPMIsupplemented with 2 mM L-glutamine, 11 mM D-glucose, 24 mM sodiumbicarbonate, 10% heat-inactivated fetal bovine serum, 100 units/mLpenicillin, 100 μg/mL streptomycin, and 0.25 μg/mL amphotericin B. MCF7was cultured in DMEM supplemented with 4 mM L-glutamine, 25 mMD-glucose, 44 mM sodium bicarbonate, 10% heat-inactivated fetal bovineserum, 100 units/mL penicillin, 100 μg/mL streptomycin, and 0.25 μg/mLamphotericin B. Both cell lines were grown at 37° C. in an atmosphere of20% O₂ and 5% CO₂. Stock cultures were maintained in continuouslyexponential growth by weekly passage of the appropriate number of cellsfollowing trypsinization with 0.25% Trypsin-EDTA (0.9 mM) in PBS.

Fluorescence microscopy: MDA-MB-468 or MCF7 cells (1×10⁵) were plated onNunc Lab-Tek® II chambered #1.5 German borosilicate coverglass (ThermoFisher Scientific, Inc.) and allowed to adhere overnight. Cells wereincubated with 100 nM Panitumumab-13 for 2 h, washed twice with PBS,incubated with 1 μM Hoechst 33342 for 0.5 h, washed twice with PBS, andimaged. Fluorescence microscopy was performed using an Evos® FL AutoImaging System (Life Technologies) at 40× magnification using acoverslip-corrected plan fluorite objective. Near-IR fluorescence wasimaged using a Cy7 LED light cube (710/40 nm excitation, 775/46 nmemission) and Hoechst using a DAPI LED light cube (357/44 nm excitation,447/60 nm emission). Image processing was conducted with ImageJ 1.49f.

Flow cytometry: MDA-MB-468 or MCF7 cells (1×10⁶) were seeded into 6-wellplates and allowed to adhere overnight. Cells were incubated with 100 nMPanitumumab-13 for 2 h and washed twice with PBS. Flow cytometricanalysis for near-IR fluorescence signal was performed at the CCR FlowCytometry Core (NCI-Frederick) using a BD LSRII Fortessa analyzeroperating a laser line at 647 nm. Data processing was conducted withFlowJo vX.0.7.

Localization characteristic of the labeled antibody was only observed inHER1+ cells by fluorescence microscopy using a standard Cy7 filter set(λ_(ex)=710 nm, λ_(em)=775 nm, FIG. 10). The efficient cellular labelingof the fluorophore-antibody conjugate was also confirmed usingfluorescence-activated cell sorting (FACS) (FIG. 11). These resultsindicated that 13 and other fluorophores disclosed herein are likely tobe suitable for a range of near-IR fluorescence applications.

Example 7 Folate Labeling

Compound 13 was converted to its NHS-ester (TSTU, DMF, 35° C.) asdescribed in Example 6, and then reacted with folate-EDA(N-(2-aminoethyl) folic acid) to provide a folate conjugate. Theconjugate was incubated with HeLa cells that were conditioned tooverexpress the folate receptor. FIG. 12A is a fluorescence image of thecells stained with Hoechst 33342. FIG. 12B is a fluorescence image ofthe cells incubated with a conjugate of folate and an exemplaryC4′-alkyl-ether heptamethine cyanine fluorophore (Folate-Cy7). FIG. 12Cis a differential interference contrast (DIC) image of the cells. FIG.12D is a merged image of the images in FIGS. 12B and 12C. Fluorescentstaining was only observed in the cells treated with the compound13-folate conjugate (FIGS. 12B, 12D). No fluorescence was seen when thecells were incubated with non-conjugated compound 13 (FIG. 12A).

Example 8 In Vivo Administration and Fluorescence-Guided Surgery

Embodiments of the disclosed C4′-alkyl-ether heptamethine cyaninefluorophores may be used for in vivo detection of target cells, such ascancer cells. In one example, a C4′-alkyl-ether heptamethine cyaninefluorophore, such as compound 13, is conjugated with an antibody capableof recognizing and binding to an antigen on a target cell. A suitablepharmaceutical composition comprising the fluorophore-antibody conjugateis administered to a subject having the target cells. For example, thepharmaceutical composition is injected into a subject with a tumorhaving an antigen to which the antibody is capable of binding. Thesubject may be a mouse with a xenograft. The tumor is irradiated withnear-IR radiation and an image may be obtained. Fluorescence indicatespresence of fluorophore-antibody conjugate bound to tumor cells. Thetumor may be excised using the detected fluorescence for guidance.Sufficient tissue is excised to provide non-fluorescent marginssurrounding the excision, indicating that the entire tumor has beenremoved.

Example 9 Cell Identification in a Tissue Sample

A C4′-alkyl-ether heptamethine cyanine fluorophore is coupled with anantibody capable of directly or indirectly recognizing and binding to anantigen on a target cell. Indirect recognition and binding is performedby first binding an anti-antigen antibody (e.g., a monoclonal antibody)to the antigen, and subsequently adding a fluorophore-antibody conjugatewhere the conjugate comprises an anti-antibody antibody (e.g., apolyclonal antibody). A tissue sample is incubated with thefluorophore-antibody conjugate under conditions sufficient to providebinding of the antibody if the antigen is present. Excess, unboundfluorophore-antibody conjugate is washed off the tissue sample. Thetissue sample is irradiated with near-infrared radiation and viewedunder conditions sufficient to visualize fluorescence emitted by thefluorophore. Fluorescence indicates presence of the antigen on thetarget cell.

Example 10 Drug Imaging

A C4′-alkyl-ether heptamethine cyanine fluorophore comprises a drug ofinterest. The drug is injected into a subject at a desired location.Presence of the drug at the desired location is verified with near-IRfluorescence imaging. If the drug preferentially locates in a particularlocation or tissue type after injection, a period of time is allowed toelapse before performing near-IR fluorescence imaging of the expectedlocation or tissue type to visualize the drug location. Excretion of thedrug, e.g., through the subject's urine, may be monitored by monitoringfluorescence of the urine over time to detect excretion of thedrug-containing fluorophore.

Example 11 In Vivo Imaging

The epidermal growth factor receptor (EGFR, HER1, c-ErbB-1) is atransmembrane glycoprotein that is a member of a subfamily of type Ireceptor tyrosine kinases including EGFR, HER2, HER3, and HER4. The EGFRis constitutively expressed in many normal epithelial tissues, includingthe skin and hair follicle. Expression of EGFR is also detected in manyhuman cancers including those of the head and neck, colon, and rectum.Panitumumab binds specifically to the EGFR on both normal and tumorcells, and competitively inhibits the binding of ligands for epidermalgrowth factor (EGF). In vitro assays and in vivo animal studies haveshown that binding of Panitumumab to the EGFR blocks phosphorylation andactivation of receptor-associated kinases, resulting in inhibition ofcell growth, induction of apoptosis, and decreased matrixmetalloproteinase and vascular endothelial growth factor production.Overexpression of this oncogene is associated with poor prognosis andaggressive tumor attributes.

A conjugate of 13 and panitumumab will be investigated in in vivooptical imaging experiments in mouse models of cancer. Animal studiesare an important step in evaluating embodiments of the disclosedC4′-alkyl-ether heptamethine cyanine fluorophores for beneficialproperties that might enable ultimate clinical application.

Mice are the preferred species for tumor imaging, because murine cancermodels most closely represent human cancers. Nude mice and cell lines(MDA-MB-231 (High-expressing), MDA-MB-468 (medium-expressing), andBT-474 (low-expressing)) will be obtained from approved sources.

Cells will be obtained from an approved source (tumor repository). Cellculturing, harvesting, and preparation will be performed. Subcutaneousinjections of up to 1×10⁶ cells in 100 μl of Hanks Balanced SaltSolution in both axillary regions will be performed. Fluorescenceimaging will be initiated when the xenograft tumor reaches approximately4-6 mm in diameter.

-   -   Imaging:    -   a) # of animals per group to provide statistically significant        data: 5;    -   b) # groups per study: 6 (3 cell lines×3 imaging agents);    -   c) 20% additional to allow for tumor incorporation variability.

Appropriate animal model (3 xenograft tumor bearing animals)×6 animalsper group×1 gender (female)×3 imaging agents: 54 animals.

Biodistribution (non-tumor bearing mice; Athymic nu/nu):

-   -   a) # of animals per time-point to provide statistically        significant data: 5 animals per time-point;    -   b) # of time-points per study: 7;    -   c) # imaging agents: 3;    -   d) # gender: 1 (female).

Two animals per time-point×7 time-points×1 gender (female and male)×3imaging agents: 42 mice.

In view of the many possible embodiments to which the principles of thedisclosed invention may be applied, it should be recognized that theillustrated embodiments are only preferred examples of the invention andshould not be taken as limiting the scope of the invention. Rather, thescope of the invention is defined by the following claims. We thereforeclaim as our invention all that comes within the scope and spirit ofthese claims.

We claim:
 1. A compound, or a pharmaceutically acceptable salt thereof,of formula I:

wherein m is 2 or 3; n is 2; R¹ is CR^(a) ₂where each R^(a)independently is H or halo; R² is optionally substituted alkyl oroptionally substituted heteroalkyl; R³ is a maleimidyl-containing group,a succinimidyl-containing group, optionally substituted alkoxy,optionally substituted alkyl carbonyl, optionally substituted alkoxycarbonyl, a drug, or a biomolecule-containing group; R⁴ and R⁹independently are H, optionally substituted alkyl, optionallysubstituted amino, or a sulfonate-containing group; R⁷ and R¹²independently are H, a sulfonate-containing group, or a trialkyl aminogroup; R¹⁴ to R¹⁷ independently are alkyl; and Z is a monatomic orpolyatomic ion having a charge sufficient to provide a neutral compound.2. The compound of claim 1, wherein: (i) R² is methyl; or (ii) R¹⁴ toR¹⁷ are methyl; or (iii) both (i) and (ii).
 3. The compound of claim 1,wherein R³ is a biomolecule-containing group.
 4. The compound of claim3, wherein the biomolecule is an antibody, a peptide, a protein, anamino acid, a nucleoside, a nucleotide, a nucleic acid, anoligonucleotide, a carbohydrate, a lipid, a hapten, or a receptorligand.
 5. The compound of claim 1, wherein R³ is a drug.
 6. Thecompound of claim 1, wherein R³ is

where p is 1, 2, 3, 4, or 5, and R^(b) is a biomolecule.
 7. The compoundof claim 6, wherein R^(b) is an antibody, a peptide, a protein, an aminoacid, a nucleic acid, nucleotide, an oligonucleotide, a lipid, a hapten,or a receptor ligand.
 8. The compound of claim 1, wherein: R⁴ and R⁹ aren-propyl or —(CH₂)₄SO₃ ⁻; and R⁷ and R¹² are hydrogen or —SO₃ ⁻Na⁺. 9.The compound of claim 8, having a structure according to formula II:


10. The compound of claim 9, wherein: R³ is

 where p is 1, 2, 3, 4, or 5, and R^(b) is a biomolecule.
 11. Thecompound of claim 10, wherein R^(b) is an antibody.
 12. The compound ofclaim 1, wherein the compound is:


13. The compound of claim 1, wherein R³ comprises folate.
 14. A method,comprising: contacting a biological sample with a compound of claim 1;irradiating the biological sample by application of light having awavelength or range of wavelengths in the near-infrared range; anddetecting fluorescence of the irradiated biological sample, whereinfluorescence indicates presence of the compound in the biologicalsample.
 15. The method of claim 14, wherein contacting the biologicalsample with the compound is performed in vivo by administering thecompound to a subject.
 16. The method of claim 15, wherein the compoundcomprises a biomolecule capable of binding to a target suspected ofbeing present within the biological sample and fluorescence indicatesthe target is present in the biological sample.
 17. The method of claim16, wherein the target is an antigen, and the compound comprises anantibody capable of recognizing and binding to the antigen.
 18. Themethod of claim 16, wherein: irradiating the biological sample comprisesirradiating a target area of the subject with near-infrared radiation;and detecting fluorescence comprises obtaining an image of theirradiated target area, wherein fluorescence in the image indicatespresence of the target in the target area.
 19. The method of claim 16,wherein the target is a tumor and the target area is an area in whichthe tumor is located.
 20. The method of claim 15, wherein the compoundcomprises a drug, the biological sample is a bodily fluid or tissue, anddetecting fluorescence of the irradiated biological sample indicatespresence of the drug in the biological sample.